Scarecrow gene, promotor and uses thereof

ABSTRACT

The structure and function of a regulatory gene, SCARECROW (SCR), is described. The SCR gene is expressed specifically in root progenitor tissues of embryos, and in roots and stems of seedlings and plants. SCR expression controls cell division of certain cell types in roots and affects the organization of root and stem tissues, and affects gravitropism of aerial structures. The invention relates to the SCARECROW (SCR) gene, SCR gene products, (including but not limited to transcriptional products such as mRNAs, antisense, and ribozyme molecules, and translational products such the SCR protein, polypeptides, peptides and fusion proteins related thereto), antibodies to SCR gene products, SCR promoters and regulatory regions and the use of the foregoing to improve agronomically valuable plants.

[0001] This application is a continuation-in-part of application ser No.08/638,617, filed Apr. 26, 1996, the disclosure of which is incorporatedby reference in its entirety.

[0002] This invention was made with government support under grantnumber: GM43778 awarded by the National Institute of Health. Thegovernment may have certain rights in the invention.

1. INTRODUCTION

[0003] The present invention generally relates to the SCARECROW (SCR)gene family and their promoters. The invention more particularly relatesto ectopic expression of members of the SCARECROW gene family intransgenic plants to artificially modify plant structures. The inventionalso relates to utilization of SCARECROW promoter for tissue and organspecific expression of heterologous gene products. cl 2. BACKGROUND OFTHE INVENTION

[0004] Asymmetric cell divisions, in which a cell divides to give twodaughters with different fates, play an important role in thedevelopment of all multicellular organisms. In plants, because there isno cell migration, the regulation of asymmetric cell divisions is ofheightened importance in determining organ morphology. In contrast toanimal embryogenesis, most plant organs are not formed duringembryogenesis. Rather, cells that form the apical meristems are setaside at the shoot and root poles. These reservoirs of stem cells areconsidered to be the source of all post-embryonic organ development inplants. A fundamental question in developmental biology is how meristemsfunction to generate plant organs.

[0005] 2.1. Root Development

[0006] Root organization is established during embryogenesis. Thisorganization is propagated during postembryonic development by the rootmeristem. Following germination, the development of the postembryonicroot is a continuous process, a series of initials or stem cellscontinuously divide to perpetuate the pattern established in theembryonic root (Steeves & Sussex, 1972, Patterns in Plant Development,Englewood Cliffs, N.J.: Prentice-Hall, Inc.).

[0007] Due to the organization of the Arabidopsis root it is possible tofollow the fate of cells from the meristem to maturity and identify theprogenitors of each cell type (Dolan et al., 1993, Development119:71-84). The Arabidopsis root is a relatively simple and wellcharacterized organ. The radial organization of the mature tissues inthe Arabidopsis root has been likened to tree rings with the epidermis,cortex, endodermis and pericycle forming radially symmetric cell layersthat surround the vascular cylinder (FIG. 1A). See also Dolan et al.,1993, Development 119:71-84. These mature tissues are derived from foursets of stem cells or initials: i) the columella root cap initial; ii)the pericycle/vascular initial; iii) the epidermal/lateral root capinitial; and iv) the cortex/endodermal initial (Dolan et al., 1993,Development 119:71-84). It has been shown that these initials undergoasymmetric divisions (Scheres et al., 1995, Development 121:53-62). Thecortex/endodermal initial, for example, first divides anticlinally (in atransverse orientation) (FIG. 1B). This asymmetric division producesanother initial and a daughter cell. The daughter cell, in turn, expandsand then divides periclinally (in the longitudinal orientation) (FIG.1B). This second asymmetric division produces the progenitors of theendodermis and the cortex cell lineages (FIG. 1B).

[0008] 2.2. Genes Regulating Root Structure

[0009] Mutations that disrupt the asymmetric divisions of thecortex/endodermal initial have been identified and characterized (Benfeyet al., 1993, Development 119:57-70; Scheres et al., 1995, Development121:53-62). short-root (shr) and scarecrow (scr) mutants are missing acell layer between the epidermis and the pericycle. In both types ofmutants the cortex/endodermal initial divides anticlinally, but thesubsequent periclinal division that increases the number of cell layersdoes not take place (Benfey et al., 1993, Development 119:57-70; Schereset al., 1995, Development 121:53-62). The defect is first apparent inthe embryo and it extends throughout the entire embryonic axis whichincludes the embryonic root and hypocotyl (Scheres et al., 1995,Development 121:53-62). This is also true for the other radialorganization mutants characterized to date, suggesting that radialpatterning that occurs during embryonic development may influence thepost-embryonic pattern generated by the meristematic initials (Schereset al., 1995, Development 121:53-62).

[0010] Characterization of the mutant cell layer in shr indicated thattwo endodermal-specific markers were absent (Benfey et al., 1993,Development 119:57-70). This provided evidence that the wild-type SHRgene may be involved in specification of endodermis identity.

[0011] 2.3. Geotropism

[0012] In plants, the capacity for gravitropism has been correlated withthe presence of amyloplast sedimentation. See, e.g., Volkmann andSievers, 1979, Encyclopedia Plant Physiol., N.S. vol 7, pp. 573-600;Sack, 1991, Intern. Rev. Cytol. 127:193-252; Bjorkmann, 1992, Adv. SpaceRes. 12:195-201; Poff et al., in The Physiology of Tropisms, Meyerowitz& Somerville (eds); Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1994) pp. 639-664; Barlow, 1995, Plant Cell Environ. 18:951-962.Amyloplast sedimentation only occurs in cells in specific locations atdistinct developmental stages. That is, when and where sedimentationoccurs is precisely regulated (Sack, 1991, Intern. Rev. Cytol.127:193-252). In roots, amyloplast sedimentation only occurs in thecentral (columella) cells of the rootcap; as these cells mature intoperipheral cap cells, the amyloplasts no longer sediment (Sack & Kiss,1989, Amer. J. Bot. 76:454-464; Sievers & Braun, in The Root Cap:Structure and Function, Wassail et al. (eds.), New York: M. Dekker(1996) pp. 31-49). In stems of many plants, including Arabidopsis,amyloplast sedimentation occurs in the starch sheath (endodermis)especially in elongating regions of the stem (von Guttenberg, DiePhysiologischen Scheiden, Handbuch der Pflanzenanatomie; K. Linsbauer(ed.), Berlin: Gebruder Borntraeger, vol. 5 (1943) p. 217; Sack, 1987,Can. J. Bot. 65:1514-1519; Sack, 15 1991, Intern. Rev. Cytol.127:193-252; Caspar & Pickard, 1989, Planta 177:185-197; Volkmann etal., 1993, J. Pl. Physiol. 142:710-6).

[0013] Gravitropic mutants have been studied for evidence that provesthe role of amyloplast sedimentation in gravity sensing. However, manygravitropic mutations affect downstream events such as auxin sensitivityor metabolism (Masson, 1995, BioEssays 17:119-127). Other mutations seemto affect gene products that process information from gravity sensing.For example, the lazy mutants of higher plants and comparable mutants inmosses can clearly sense and respond to gravity, but the mutationsreverse the normal polarity of the gravitropic response (Gaiser & Lomax,1993, Plant Physiol. 102:339-344; Jenkins et al., 1986, Plant CellEnviron 9:637-644). Other mutations appear to affect gravitropism ofspecific organs. For example, sgr mutants have defective shootgravitropism (Fukaki et al., 1996, Plant Physiol. 110:933-943; Fukaki etal., 1996, Plant Physiol. 110:945-955; Fukaki et al., 1996, Plant Res.109:129-137).

[0014] Citation or identification of any reference herein shall not beconstrued as an admission that such reference is available as prior artto the present invention.

3. SUMMARY OF THE INVENTION

[0015] The structure and function of a regulatory gene, SCARECROW (SCR),is described. The SCR gene is expressed specifically in root progenitortissues of embryos, and in certain tissues of roots and stems. SCRexpression controls cell division of certain cell types in roots, andaffects the organization of root and stem. The invention relates to theSCARECROW (SCR) gene (which encompasses the Arabidopsis SCR gene and itsorthologs and paralogs), SCR gene products, (including but not limitedto transcriptional products such as mRNAs, antisense and ribozymemolecules, and translational products such as the SCR protein,polypeptides, peptides and fusion proteins related thereto), antibodiesto SCR gene products, SCR regulatory regions and the use of theforegoing to improve agronomically valuable plants.

[0016] The invention is based, in part, on the discovery, identificationand cloning of the gene responsible for the scarecrow phenotype. Incontrast to the prevailing view that the SCR gene was likely to beinvolved in the specification of endodermis, the inventors havedetermined that the mutant cell layer in roots of scr mutants hasdifferentiated characteristics of both cortex and endodermis. This isconsistent with a role for SCR in the regulation of the asymmetric celldivision rather than in specification of the identity of either cortexor endodermis. The inventors have also determined that SCR expressionaffects the gravitropism of plant aerial structures such as the stem.

[0017] One aspect of the invention relates to the heterologousexpression of SCR genes and related nucleotide sequences, andspecifically the Arabidopsis SCR genes, in stably transformed higherplant species. Modulation of SCR expression levels can be used toadvantageously modify root and aerial structures of transgenic plantsand enhance the agronomic properties of such plants.

[0018] Another aspect of the invention relates to the use of promotersof SCR genes, and specifically the use of Arabidopsis SCR promoter tocontrol the expression of protein and RNA products in plants. Plant SCRpromoters have a variety of uses, including but not limited toexpressing heterologous genes in the embryo, root, root nodule, and stemof transformed plants.

[0019] The invention is illustrated by working examples described infrawhich demonstrate the isolation of the Arabidopsis SCR gene usinginsertion mutagenesis. More specifically, T-DNA tagging of genomic andcDNA clones of the Arabidopsis SCR gene are described. Additionalworking examples include the isolation of SCR sequences from plantgenomes using PCR amplification in combination with screening of genomiclibraries, and heterologous gene expression in transgenic plants usingSCR promoter expression constructs.

[0020] Structural analysis of the deduced amino acid sequence ofArabidopsis SCR protein indicates that SCR encodes a transcriptionfactor. Northern analysis, in situ hybridization analysis and enhancertrap analysis show highly localized expression of Arabidopsis SCR inembryos and roots. Genetic analysis shows SCR expression also affectsgravitropism of aerial structures (e.g., stems). This indicates that SCRis also expressed in those structures.

[0021] Computer analysis of the deduced amino acid sequence ofArabidopsis SCR protein with those of Expressed Sequence Tag (EST)sequences in GenBank reveals the existence of at least thirteen SCRgenes in Arabidopsis, one SCR gene in maize, four SCR genes in rice, andone SCR gene in Brassica. A further aspect of the invention relates tothe use of such EST sequences to obtain larger and/or complete clones ofthe corresponding SCR gene.

[0022] The various embodiments of the claimed invention presented hereinare by the way of illustration and are not meant to limit the invention.

[0023] 3.1. Definitions

[0024] As used herein, the terms listed below will have the meaningsindicated. 35S = cauliflower mosaic virus promoter for the 35Stranscript cDNA = complementary DNA cis-regulatory = A promoter sequence5′ upstream of the TATA element box that confers specific regulatoryresponse to a promoter containing such an element. A promoter maycontain one or more cis- regulatory elements, each responsible for aparticular regulatory response coding = sequence that encodes a completeor partial sequence gene product (e.g., a complete protein or a fragmentthereof) DNA = deoxyribonucleic acid EST = expression tagged functional= a functional portion of a promoter is any portion portion of apromoter that is capable of causing transcription of a linked genesequence, e.g., a truncated promoter gene = a gene construct comprisinga promoter fusion operably linked to a heterologous gene, wherein saidpromoter controls the transcription of the heterologous gene gene = theRNA or protein encoded by a gene sequence product gene = sequence thatencodes a complete gene product sequence (e.g., a complete protein) GUS= l,3-β-Glucuronidase gDNA = genomic DNA heterologous = In the contextof gene constructs, a gene heterologous gene means that the gene islinked to a promoter that said gene is not naturally linked to. Theheterologous gene may or may not be from the organism contributing saidpromoter. The heterologous gene may encode messenger RNA (mRNA) ,antisense RNA or ribozymes homologous = a native promoter of a gene thatselectively promoter hybridizes to the sequence of a SCR gene describedherein mRNA = messenger RNA operably = A linkage between a promoter andgene sequence linked such that the transcription of said gene sequenceis controlled by said promoter ortholog = related gene in a differentplant (e.g., maize ZCARECROW gene is an ortholog of the Arabidopsis SCRgene) paralog = related gene in the same plant (e.g., Arabidopsis SRPa1is a paralog of Arabidopsis SCR gene) RNA = ribonucleic acid RNase =ribonuclease SCR = SCARECROW gene or gene product, encompasses (italic)SCR and ZCR genes and their orthologs and paralogs SCR = SCARECROWprotein scr = scarecrow mutant (e.g., scr1) (lower case) ZCR = maizeZCARECROW gene, a paralog of, for example, the Arabidopsis SCR gene

[0025] SCR protein means a protein containing sequences or a domainsubstantially similar to one or more motifs (i.e., Motif I-VI),preferably MOTIF III (VHIID), of Arabidopsis SCR protein as shown inFIGS. 13A-F and FIGS. 15A-S. SCR proteins include SCR ortholog andparalog proteins having the structure and activities described herein.

[0026] SCR polypeptides and peptides include deleted or truncated formsof the SCR protein, and fragments corresponding to the SCR motifsdescribed herein.

[0027] SCR fusion proteins encompass proteins in which the SCR proteinor an SCR polypeptide or peptide is fused to a heterologous protein,polypeptide or peptide.

[0028] SCR gene, nucleotides or coding sequences means nucleotides,e.g., gDNA or cDNA encoding SCR protein, SCR polypeptides or peptides,or SCR fusion proteins.

[0029] SCR gene products include transcriptional products such as mRNAs,antisense and ribozyme molecules, as well as translational products ofthe SCR nucleotides described herein including but not limited to theSCR protein, polypeptides, peptides and/or SCR fusion proteins.

[0030] SCR promoter means the regulatory region native to the SCR genein a variety of species, which promotes the organ and tissue specificpattern of SCR expression described herein.

4. BRIEF DESCRIPTION OF THE FIGURES

[0031] FIGS. 1A-B. Schematic of Arabidopsis root anatomy. FIG. 1A.Transverse section showing the four tissues, epidermis, cortex,endodermis and pericycle that surround the vascular tissue. In thelongitudinal section, the epidermal/lateral root cap initials and thecortex/endodermal initials are shown at the base of their respectivecell files. FIG. 1B. Schematic of division pattern of thecortex/endodermal initial. The initial expands then divides anticlinallyto reproduce itself and a daughter cell. The daughter then dividespericlinally to produce the progenitors of the endodermis and cortexcell lineages. Abbreviations: C, cortex; Da, daughter cell; E,endodermis; In, initial.

[0032] FIGS. 2A-F. Phenotype of scr mutant plants. FIG. 2A. Shown leftto right are 12-day scr-2,-scr-1 and wild-type seedlings grownvertically on nutrient agar medium. FIG. 2B. 21-day scr-2 mutant plantsin soil. FIG. 2C. Transverse section through primary root of 7-dayscr-2. FIG. 2D. Transverse section through primary root of 7-daywild-type (WT). FIG. 2E. Transverse section through lateral root of12-day scr-1 mutant seedling. FIG. 2F. Transverse section through rootregenerated from scr-1 callus. Bar, 50 μm. Abbreviations: C, cortex; En,endodermis; Ep, epidermis; M, mutant cell layer; P, pericycle; V,vascular tissue.

[0033] FIGS. 3A-F. Characterization of the cellular identity of themutant cell layer. FIG. 3A. Endodermis-specific Casparian band stainingof transverse sections through the primary root of 7-day scr-1 mutant.(Note: the histochemical stain also reveals xylem cells in the vascularcylinder.) FIG. 3B. Casparian band staining of transverse sectionsthrough the primary root of 7-day wild-type (WT). FIG. 3C.Immunostaining with the endodermis (and a subset of vascular tissue)specific JIM13 monoclonal antibodies on transverse root sections ofscr-2 mutant. FIG. 3D. Immunostaining with JIM13 monoclonal antibodieson transverse root sections of WT. FIG. 3E. Immunostaining with the JIM7monoclonal antibody that stains all cell walls on transverse rootsections of scr-2 mutant. FIG. 3F. Immunostaining with JIM7 monoclonalantibodies on transverse root sections of WT. Bar, 25 μm. Abbreviationsare same as those for description of FIGS. 2A-2F and: Ca, casparianstrip.

[0034] FIGS. 4A-F. Immunostaining. FIG. 4A. Immunostaining with thecortex (and epidermis) specific CCRC-M2 monoclonal antibodies ontransverse root sections of scr-1 mutant. FIG. 4B. Immunostaining withCCRC-M2 antibodies on transverse root sections of scr-2 mutant. FIG. 3C.Immunostaining with CCRC-M2 antibodies on transverse root sections ofwild-type (WT). FIG. 4D. Immunostaining with the CCRC-M1 monoclonalantibodies (specific to a cell wall epitope found on all cells) ontransverse root sections of scr-1. FIG. 4E. Immunostaining with CCRC-M1antibodies on transverse root sections of scr-2. FIG. 4F. Immunostainingwith CCRC-M1 antibodies on transverse root sections of WT. Bar, 30 μm.Abbreviations are same as those for description of FIGS. 2A-2F.

[0035]FIG. 5A-E. Structure of the Arabidopsis SCARECROW gene. FIG. 5A.Nucleic acid sequence and deduced amino acid sequence of the ArabidopsisSCR genomic region (SEQ ID NO:1) and (SEQ ID NO:2), respectively.Regulatory sequences including: (i) TATA box, (ii) ATG start codon, and(iii) potential polyadenylation sequence are underlined. Within thededuced amino acid sequence homopolymeric repeats are underlined. FIG.5B. Schematic diagram of genomic clone indicating possible functionalmotifs, T-DNA insertion sites and subclones used as probes.Abbreviations: Q,S,P,T, region with homopolymeric repeats of these aminoacids; b, region with similarity to the basic region of bZIP factors; Iand II, regions with leucine heptad repeats; E, acidic region. FIG. 5C.Comparison of the charged region found in Arabidopsis SCR protein withthat found in bZIP transcription factors, SCR bZIP-like domain (SEQ IDNO:3), GCN4 (SEQ ID NO:4), TGA1 (SEQ ID NO:5), C-Fos (SEQ ID NO:6),c-JUN (SEQ ID NO:7), CREB (SEQ ID NO:8), Opaque-2 (SEQ ID NO:9), OBF2(SEQ ID NO:10), RAF-1 (SEQ ID NO:11). FIG. 5D. Translations of ESTclones encoding putative peptide having similarities to 15 the VHIIDdomain region of Arabidopsis SCR protein (SEQ ID NO:12), F13896 (SEQ IDNO:13), Z37192 (SEQ ID NO:14), and Z25645 (SEQ ID NO:15) are fromArabidopsis, T18310 (SEQ ID NO:17) is from maize and D41474 (SEQ IDNO:16) is from rice. FIG. 5E. The deduced amino acid sequence of theArabidopsis SCARECROW gene (SEQ ID NO:2).

[0036] FIGS. 6A-B. Expression of the Arabidopsis SCARECROW gene. FIG.6A. Northern blot of total RNA from wild-type siliques (Si), roots (R),leaves (L) and whole seedlings (Sd) hybridized with Arabidopsis SCRprobe a and with a probe from the Arabidopsis glutamine dehydrogenase(GDH) gene (Melo-Oliveira et al., 1996, Proc. Natl. Acad. Sci. USA93:4718-4723) as a control for RNA integrity. (GDH expression is lowerin siliques than in vegetative tissues.) The 1.6 kb band corresponds tothe GDH gene and the approximately 2.5 kb band corresponds to SCR.Ribosomal RNA is shown as a loading control. FIG. 6B. Northern blot ofArabidopsis wild-type, scr-1 and scr-2 total RNA, probed withArabidopsis SCR probe “a” corresponding to a cDNA sequence shown in FIG.5B, and with the GDH probe. In scr-2 mutant additional bands of 4.1 kband 5.0 kb were detected.

[0037] FIGS. 7A-G. In situ hybridization and enhancer trap analyses ofArabidopsis SCR expression. FIG. 7A. SCR RNA expression detected by insitu hybridization of SCR antisense probe to a longitudinal sectionthrough the root meristem. FIG. 7B. In situ hybridization of SCRantisense probe to a transverse section in the meristematic region. FIG.7C. In situ hybridization of SCR antisense probe to late torpedo stageembryo. FIG. 7D. Negative control in situ hybridization using a SCRsense probe to a longitudinal section through the root meristem. FIG.7E. GUS expression in a whole mount in the enhancer trap line, ET199 inprimary root tip. FIG. 7F. GUS expression in the ET199 line intransverse root section in the meristematic region. FIG. 7G. GUSexpression in ET199 detected in a section through the root meristem. GUSexpression is observed in the cortex/endodermal initial, and in thefirst cell in the endodermal cell lineage but not in the first cell ofthe cortex lineage. Expression in two endodermal layers is observedhigher up in the root because the section was not median at that point.Bar, 50 μm. Abbreviations are same as those in the description of FIGS.2A-2.

[0038]FIG. 8. Partial nucleotide sequence (SEQ ID NO:18) and deducedamino acid sequence (SEQ ID NO:19) of the Arabidopsis SRPa4 gene.

[0039]FIG. 9. Partial nucleotide sequence (SEQ ID NO:20) and deducedamino acid sequence (SEQ ID NO:21) of the Arabidopsis SRPa3 gene.

[0040]FIG. 10. Partial nucleotide sequence (SEQ ID NO:22) of theArabidopsis SRPa1 gene.

[0041]FIG. 11A. Nucleotide sequence (SEQ ID NO:24) and deduced aminoacid sequence (SEQ ID NO:25) of the maize Zm-Scl1 fragment.

[0042]FIG. 11B. Partial nucleotide sequence (SEQ ID NO:25) and deducedamino acid sequence (SEQ ID NO:26) of the maize SRPm1 gene (Zm-Scl2).

[0043]FIG. 12A-B. Nucleotide sequence of rice SRPo3 EST clone. FIG. 12A.Sequence of 5′ end of EST clone (SEQ ID NO:28). FIG. 12B. Sequence of 3′end of EST clone (SEQ ID NO:29).

[0044] FIGS. 13A-F. Comparison of the amino acid sequence of members ofthe SCARECROW family of genes. Conserved Motifs I through VI areindicated by dashed line above the aligned sequences. Consensussequences are shown in bold. See Table 1 for the identity and sequenceidentifier number of each of the sequences shown in this Figure.Hu-scr-1= Human SCR paralog (SEQ ID NO:40).

[0045]FIG. 14. Restriction map of the approximately 8.8 kb Eco RI insertDNA of lambda clone, t643, containing the Arabidopsis SCR gene. Thelocations of the approximately 5.6 kb HindIII-SacI fragment subcloned inplasmid LIG 1-3/SAC+MoB₂ 1SAC, and the SCR coding region are indicatedbelow the restriction map. The location of the translational initiationsite of the SCR gene is at the Nco I site at the left end of theindicated coding region. The SCR coding sequence begins at thetranslation initiation site and extends approximately 1955 nucleotidesto its right. E. coliDH5α containing plasmid pLIG1-3/SAC+MoB₂ 1SAC, hasthe ATCC accession number 98031.

[0046] FIGS. 15A-S. Comparison of the partial and complete amino acidsequences of several plant members of the SCARECROW family of genes. Theamino acid sequences are aligned in a manner that maximizes amino acidsequence similarity and identity among SCR family members. Each sequenceshown is continuous except where noted otherwise; the dots are insertedbetween two sequence segments in order to align homologous segments. “X”in the middle of a sequence indicates ambiguity in the correspondingnucleotide sequence and, possible termination of the ORF at the “X”residue site. “X” at the end of a sequence indicates termination of theORF at the “X” residue site. The numbering of the amino acid residues isshown at the bottom of each figure and is based on the Arabidopsis SCRamino acid sequence. Conserved Motifs I through VI are indicated by thevarious dashed lines above the figures. The new and old names of thefamily members are shown in FIG. 15A. The sequences of SCR, Tf1 and Tf4are of the complete SCR protein. See Table 1 for the identity and thesequence identifier number of each sequence shown in these figures.

[0047] FIGS. 16A-M. The partial nucleotide sequences of several plantmembers of the SCARECROW family of genes. “N” indicates an unknown base.See Table 1 for the identity and the sequence identifier number of eachsequence shown in these figures.

[0048]FIG. 17A. The partial nucleotide sequence (SEQ ID NO:66) of themaize ZCR gene.

[0049]FIG. 17B. The partial amino acid sequence (SEQ ID NO:67) of themaize ZCR gene. The underlined sequence shares approximately 80%sequence identity with a corresponding sequence of Arabidopsis SCRprotein.

[0050]FIG. 18. Comparison of the partial amino acid sequences of severalSCR ortholog sequences amplified from the genomes of carrot, soybean andspruce. The SRPd1 and aSRPp1 sequences each were obtained by PCRamplification using a combination of 1F and 1R primers. The SRPg1sequence was obtained by PCR amplification using a combination of 1F andWP primers. The amino acid sequences are aligned in a manner thatmaximizes amino acid sequence identity and similarity amongst thesesequences. Each sequence shown is continuous except where notedotherwise; the dashes are inserted between two sequence segments inorder to allow alignment of homologous segments. “X” in the middle of asequence indicates ambiguity in the corresponding nucleotide sequenceand, possible termination of the ORF or existence of an intron at the“X” residue site. See Table 1 for the identity and the sequenceidentifier number of each sequence shown in this figure.

[0051]FIG. 19. Comparison of promoter activities in transgenic lines androots. Panel a. A stably transformed line containing four copies of theB2 subdomain of the 35S promoter of CaMV upstream of GUS (Benfey et al.,1990). GUS is expressed in the root tip. Panel b. Roots emerging fromcallus transformed with four copies of the B2 subdomain of the 35Spromoter fused to GUS. GUS expression can be seen in the emerging roottips (arrows). Panel c. Higher magnification of a root emerging from thecallus in panel b. GUS is clearly restricted to the root tip. Themorphology of roots regenerated from calli often appears abnormal. Paneld. A transgenic plant regenerated from the calli and roots shown inpanel b. GUS expression in this plants appears to be similar to that ofthe original line shown in panel a. Panel e. ET199, a stably transformedline that contains an enhancer trapping construct with a minimalpromoter fused to the GUS coding region inserted 1 kb upstream from theSCR coding region. GUS expression is primarily in the endodermal layerof the root. Panel f. Roots emerging from calli transformed with the SCRpromoter::GUS construct. Expression of the GUS gene appears to belimited to an internal layer (arrows). Panel g. SCR promoter: :GUStransformed root in liquid culture. Roots shown in panel f were excisedand transferred to liquid cultures. GUS expression is primarily found inthe endodermal layer as in ET199. The expression of GUS in the quiescentcenter, as seen here, is also sometimes observed in ET199. Bar, 50 μm.

[0052]FIG. 20. Analysis of SCR promoter activity in the scr mutantbackground. Panel a. Roots emerging from scr calli transformed with theSCR promoter: :GUS construct. Roots regenerated from scr calli are veryshort. GUS expression appears to be limited to an internal layer of theroot (arrows). Panel b. Root regenerated from transformed scr calli andtransferred to liquid culture. The scr phenotype, a single layer betweenthe epidermis and pericycle, is easily seen. GUS expression is limitedto this mutant layer. E, Epidermis. M, Mutant Layer. P, Pericycle. Bar,50 μm.

[0053]FIG. 21. Molecular Complementation of the scr mutant. Panels a, cand e. scr transformed with the SCR promoter::GUS construct. Panels b, dand f. scr transformed with the SCR promoter::SCR coding regionconstruct. Panels a and b. Roots emerging from scr calli. Arrows pointto several very short roots among many fine root hairs in the scr callitransformed with the SCR promoter::GUS construct. In contrast, rootsfrom scr calli transformed with the SCR promoter::SCR coding regionconstruct appeared to be wild-type in length, suggesting molecularcomplementation by the transgene. Panels c and d. Transgenic roots inliquid culture. The scr roots transformed with the SCR promoter::GUSconstruct appeared short, while those transformed with the SCRpromoter::SCR coding region construct appeared of wild-type length.Panels e and f. Transverse sections through roots emerging from calli.Whereas there is only a single cell layer between the epidermis andstele in the SCR promoter::GUS transformed root, the radial organizationof the root transformed with the SCR promoter::SCR coding regionappeared identical to wild-type, with both cortex and endodermal layers.E, epidermis. M, mutant layer. C, cortex. En, Endodermis. P, Pericycle.Bar, 50 μm FIG. 22. Expression of ZCR in maize root tips. Left Panel.Expression of ZCR is in the endodermal layer and extends down throughthe region of the quiescent center. Right Panel. Higher magnificationshowing expression in a single cell layer through the quiescent center.

5. DETAILED DESCRIPTION OF THE INVENTION

[0054] The invention relates to the SCARECROW (SCR) gene, SCR geneproducts, including but not limited to transcriptional products such asmRNAs, antisense and ribozyme molecules, and translational products suchas the SCR protein, polypeptides, peptides and fusion proteins relatedthereto; antibodies to SCR gene products; SCR regulatory regions; andthe use of the foregoing to improve agronomically valuable plants.

[0055] In summary, the data described herein show the identification ofSCR, a gene involved in the regulation of a specific asymmetricdivision, in controlling gravitropic response in aerial structures, andin controlling pattern formation in roots. Sequence analysis shows thatthe SCR protein has many hallmarks of transcription factors. In situ andmarker line expression studies show that SCR is expressed in thecortex/endodermal initial of roots before asymmetric division occurs,and in quiescent center of regenerating roots. Together, these findingsindicate that SCR gene regulates key events that establish theasymmetric division that generates separate cortex and endodermal celllineages, and that affect tissue organization of roots. Theestablishment of these lineages is not required for cell differentiationto occur, because in the absence of division the resulting cell acquiresmature characteristics of both cortex and endodermal cells. However, itis possible that SCR functions to establish the polarity of the initialbefore cell division, or that it is involved in generating an externalpolarity that has an effect on asymmetric cell division.

[0056] Genetic analysis indicates that SCR expression affectsgravitropism of plant stems and hypocotyls. This indicates that SCR isalso expressed in these aerial structures of plants.

[0057] The SCR genes and promoters of the present invention have anumber of important agricultural uses. The SCR promoters of theinvention may be used in expression constructs to express desiredheterologous gene products in the embryo, root, root nodule, and starchsheath layer in stem of transgenic plants transformed with suchconstructs. For example, SCR promoters may be used to express diseaseresistance genes such as lysozymes, cecropins, maganins, or thionins foranti-bacterial protection or the pathogenesis-related (PR) proteins suchas glucanases and chitinases for anti-fungal protection. SCR promotersalso may be used to express a variety of pest resistance genes in theaforementioned plant structures and tissues. Examples of useful geneproducts for controlling nematodes or insects include Bacillusthuringiensis endotoxins, protease inhibitors, collagenases, chitinase,glucanases, lectins, and glycosidases.

[0058] Gene constructs that express or ectopically express SCR, and theSCR-suppression constructs of the invention may be used to alter theroot and/or stem structure, and the gravitropism of aerial structures oftransgenic plants. Since SCR regulates root cell divisions,overexpression of SCR can be used to increase division of certain cellsin roots and thereby form thicker and stronger roots. Thicker andstronger roots are beneficial in preventing plant lodging. Conversely,suppression of SCR expression can be used to decrease cell division inroots and thereby form thinner roots. Thinner roots are more efficientin uptake of soil nutrients. Since SCR affects gravitropism of aerialstructures, overexpression of SCR may be used to develop “straighter”transgenic plants that are less susceptible to lodging.

[0059] Further, SCR gene sequence may be used as a molecular marker fora qualitative trait, e.g., a root or gravitropism trait, in molecularbreeding of crop plants.

[0060] For purposes of clarity and not by way of limitation, theinvention is described in the subsections below in terms of (a) SCRgenes and nucleotides; (b) SCR gene products; (c) antibodies to SCR geneproducts; (d) SCR promoters and promoter elements; (e) transgenic plantswhich ectopically express SCR; (f) transgenic plants in which endogenousSCR expression is suppressed; and (g) transgenic plants in whichexpression of a transgene of interest is controlled by SCR promoter.

5.1. Scr Genes

[0061] The SCARECROW genes and nucleotide sequences of the inventioninclude: (a) a gene listed below in Table 1 (hereinafter, a genecomprising any one of the nucleotide sequences shown in FIG. 5A, FIG. 8,FIG. 9, FIG. 10, FIGS. 11A-B, FIGS. 12A-B, FIGS. 16A-M, or FIG. 17A, ora segment of such nucleotide sequences), or as contained in the clonesdescribed herein and deposited with the ATCC (see Section 13, infra);(b) nucleotide sequence that encodes a protein comprising any one of theamino acid sequences shown in FIG. 5A, FIG. SD, FIG. 5E, FIG. 8, FIG. 9,FIGS. 11A-B, FIGS. 13A-F, FIGS. 15A-S, FIG. 17B or FIG. 18 or a segmentof such amino acid sequences, or that is encoded by any one of the genesand/or nucleotide sequences listed by their sequence identifier numbersin Table 1, or any segment of such genes and/or nucleotide sequences, orcontained in any one of the clones described herein and deposited withthe ATCC (see Section 13, infra); (c) any gene comprising nucleotidesequence that hybridizes to the complement of any one of the genesand/or nucleotide sequences listed by their sequence identifier numbersin Table 1, or any segment of such genes and/or nucleotide sequences, oras contained in any one of the clones described herein and depositedwith the ATCC, under highly stringent conditions, e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO_(4,) 7% sodium dodecyl sulfate (SDS), 1mM EDTA at 65° C., and washing in 0.1xSSC/0.1% SDS at 68° C. (AusubelF.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork, at p. 2.10.3) and that encodes a gene product functionallyequivalent to SCR gene product encoded completely or partly by any oneof the genes and/or sequences listed in Table 1 or any segment of suchgenes and nucleotide sequences, or as contained in any one of the clonesdeposited with the ATCC; (d) any gene comprising nucleotide sequencethat hybridizes to the complement of any one of the sequences listed bytheir sequence identifier numbers in Table 1, or any segment of suchnucleotide sequences, or as contained in any one of the clones describedherein and deposited with the ATCC, under less stringent conditions,such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1%SDS at 42° C. (Ausubel et al., 1989, supra), and which encodes afunctionally equivalent SCR gene product; (e) any gene comprisingnucleotide sequence that hybridizes to the complement of any one of thesequences listed by their sequence identifier numbers in Table 1 or anysegment of such nucleotide sequences, or as contained in any one of theclones described herein and deposited with the ATCC, under the followinglow stringency conditions: pre-hybridization in hybridization solution(HS) containing 43% formamide, 5×SSC, 1% SDS, 10% dextran sulfate, 0.1%sarkosyl, 2% block (Genius kit, Boehringer-Mannheim), followed byhybridization overnight at 30 to 33° C. using as a probe a DNA moleculeof approximately 1.6 kb of SEQ ID NO:1 at a concentration of 20 ng/ml,followed by washing in 2×SSC/0.1% SDS two times for 15 minutes at roomtemperature and then two times at 50° C., and which encodes afunctionally equivalent SCR gene product; and/or (f) any gene comprisingnucleotide sequence that encodes a polypeptide or protein containing theconsensus sequence for SCR (i.e., MOTIF III or VHIID) shown in FIGS.13B-D or a segment of such polypeptide or protein. The partial andcomplete nucleotide and amino acid sequences of SCR genes and encodedproteins and polypeptides included in the invention are listed in Table1 below. TABLE 1 SCR ORTHOLOGS AND PARALOGS SEQ ID NOs Nucleo- Amino NewName Old Name EST Clone¹ tide³ Acid ARABIDOPSIS SRPa1 1110 Z25645/3377222 23 SRPa2 Tf4 Z34599 —  35* SRPa3 3935 Z37192/1 20 21 N96166 SRPa44818 F13896/7 18 19 SRPa5 4871 F13949 45 46 SRPa6 12398 R29793 51 52SRPa7 3635 T21627 55 56 H76979 N96767 SRPa8 Tf1 T46205 (9468) —  34*N96653 (21711) SRPa9 10964 T78186 47 48 T44774 SRPa10 11261 T76483 49 50SRPa11 18652 N37425 53 54 SRPa12 23196 W43803 57 58 W435138 AA042397SRPa13 33/08 T46008 — 41 SCR Scr N.A.²  1⁺  2* RICE SRPo1 713 D15490 —43 SRPo2 2504 D40482 — 44 D40607 D40800 D41389 SRPo3 3989 D41474 — 36SRPo4 11846 C20324 — 59 MAIZE SRPm1 18310 T18310 — 37 BRASSICA SRPb1 174H74669 — 42 CARROT SRPd1 N.A. N.A. 60 61 SOYBEAN SRPg1 N.A. N.A. 62 63SPRUCE SRPp1 N.A. N.A. 64 65

[0062] Functional equivalents of the SCR gene product include any plantgene product that regulates plant embryo or root development, or,preferably, that regulates root cell division or root tissueorganization, or affects gravitropism of plant aerial structures (e.g.,stems and hypocotyls). Functional equivalents of the SCR gene productinclude naturally occurring SCR gene products, and mutant SCR geneproducts, whether naturally occurring or engineered.

[0063] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements ofthe nucleotide sequences (a) through (f), in the first paragraph of thissection. Such hybridization conditions may be highly stringent, lesshighly stringent, or low stringency as described above. In instanceswherein the nucleic acid molecules are oligonucleotides (“oligos”),highly stringent conditions may refer, e.g., to washing in 6×SSC/0.05%sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may act as SCR antisense molecules, useful,for example, in SCR gene regulation and/or as antisense primers inamplification reactions of SCR gene and/or nucleic acid sequences.Further, such sequences may be used as part of ribozyme and/or triplehelix sequences, also useful for SCR gene regulation. Still further,such molecules may be used as components in probing methods whereby thepresence of a SCARECROW allele may be detected.

[0064] The invention also includes nucleic acid molecules, preferablyDNA molecules, which are amplified using the polymerase chain reactionunder conditions described in Section 5.1.1., infra, and that encode agene product functionally equivalent to a SCR gene product encoded byany one of the genes and sequences listed in Table 1 or as contained inany one of the clones described herein and deposited with the ATCC.

[0065] The invention also encompasses (a) DNA vectors that contain anyof the foregoing gene and/or coding sequences and/or their complements(i.e., antisense or ribozyme molecules); (b) DNA expression vectors thatcontain any of the foregoing gene and/or coding sequences operativelyassociated with a regulatory element that directs the expression of thegene and/or coding sequences; and (c) genetically engineered host cellsthat contain any of the foregoing gene and/or coding sequencesoperatively associated with a regulatory element that directs theexpression of the gene and/or coding sequences in the host cell. As usedherein, regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

[0066] The invention also encompasses nucleotide sequences that encodemutant SCR gene products, peptide fragments of the SCR gene product,truncated SCR gene products, and SCR fusion proteins. These geneproducts include, but are not limited to, nucleotide sequences encodingmutant SCR gene products; polypeptides or peptides corresponding to oneor more of the Motifs I-VI as shown in FIGS. 13A-F and FIGS. 15A-S, orthe bZIP, VHIID, or leucine heptad domains of the SCR, or portions ofthese motifs and domains; truncated SCR gene products in which one ormore of the motifs or domains is deleted, e.g., a truncated,nonfunctional SCR lacking all or a portion of the Motifs I-VI as shownin FIGS. 13A-F and FIGS. 15A-S, or the bZIP, VHIID, or leucine heptaddomains of the SCR. Nucleotides encoding fusion proteins may include butare not limited to full length SCR, truncated SCR or peptide fragmentsof SCR fused to an unrelated protein or peptide, such as for example, anenzyme, fluorescent protein, or luminescent protein which can be used asa marker.

[0067] In particular, the invention includes, for example, fragments ofSCR genes encoding one or more of the following domains as shown in FIG.5E: amino acids 1-264, 265-283, 287-316, 410-473, 436-473, and 473-653.

[0068] In addition to the gene and/or coding sequences described above,homologous SCR genes, and other genes related by DNA sequence, may beidentified and may be readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Morespecifically, such homologs include, for example, paralogs (i.e.,members of the SCR gene family occurring in the same plant) as well asorthologs (i.e., members of the SCR gene family which occur in adifferent plant species) of the Arabidopsis SCR gene.

[0069] A specific embodiment of a SCR gene and coding sequence of theinvention is Arabidopsis SCR (FIGS. 5A and 5E). Other specificembodiments include the various SCR genes and coding sequences listed inTable 1, supra.

[0070] Methods for isolating SCR genes and coding sequences aredescribed in detail in Section 5.2, below.

[0071] SCR genes share substantial amino acid sequence similarities atthe protein level and nucleotide sequence similarities in their encodinggenes. The term “substantially similar” or “substantial similarity” whenused herein with respect to two amino acid sequences means that the twosequences have at least 75% identical residues, preferably at least 85%identical residues and most preferably at least 95% identical residues.The same term when used herein with respect to two nucleotide sequencesmeans that the two sequences have at least 70% identical residues,preferably at least 85% identical residues and most preferably at least95% identical residues. Determining whether two sequences aresubstantially similar may be carried out using any methodologies knownto one skilled in the art, preferably using computer assisted analysis.For example, the alignments showed herein were initially accomplished bya BLAST search (NCBI using the BLAST network server). The finalalignments of SCR family members were done manually.

[0072] Moreover, SCR genes show highly localized expression in embryosand, particularly, roots. Such expression patterns may be ascertained byNorthern hybridizations and in situ hybridizations using antisenseprobes.

[0073] 5.1.1. ISOLATION OF SCR GENES

[0074] The following methods can be used to obtain SCR genes and codingsequences from a wide variety of plants, including but not limited toArabidopsis thaliana, Zea mays, Nicotiana tabacum, Daucus carota, Oryza,Glycine max, Lemna gibba, and Picea abies.

[0075] Nucleotide sequences encoding an SCR gene or a portion thereofmay be obtained by PCR amplification of plant genomic DNA or cDNA.Useful cDNA sources include “free” cDNA preparations (i.e., the productsof cDNA synthesis) and cloned cDNA in cDNA libraries. Root cDNApreparations or libraries are particularly preferred.

[0076] The amplification may use, as the 5′-primer (i.e., forwardprimer), a degenerate oligonucleotide that corresponds to a segment of aknown SCR amino acid sequence, preferably from the amino-terminalregion. The 3′-primer (i.e., reverse primer) may be a degenerateoligonucleotide that corresponds to a distal segment of the same knownSCR amino acid sequence (i.e., carboxyl to the sequence that correspondsto the 5′-primer). For example, the amino acid sequence of theArabidopsis SCR protein (SEQ ID NO:2) may be used to design useful 5′and 3′ primers. Preferably, the primers corresponds to segments in theMotif III or VHIID domain of SCR protein (see FIGS. 13B-D and FIGS.15K-L). The sequence of the optimal degenerate oligonucleotide probecorresponding to a known amino acid sequence may be determined bystandard algorithms known in the art. See for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Vol 2 (1989).

[0077] Further, for amplification from CDNA sources, the 3′-primer maybe an oligonucleotide comprising an 3′ oligo(dT) sequence. Theamplification may also use as primers nucleotide sequences of SCR genesor coding sequences (e.g., any one of the scr sequences and ESTsequences listed in Table 1).

[0078] PCR amplification can be carried out, e.g., by use of aPerkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp™). Onecan choose to synthesize several different degenerate primers for use inthe PCR reactions. It is also possible to vary the stringency ofhybridization conditions used in priming the PCR reactions, to allow forgreater or lesser degrees of nucleotide sequence similarity between thedegenerate primers and the corresponding sequences in the cDNA library.One of ordinary skill in the art will know that the appropriateamplification conditions and parameters depend, in part, on the lengthand base composition of the primers and that such conditions may bedetermined using standard formulae. Protocols for executing all PCRprocedures discussed herein are well known to those skilled in the art,and may be found in references such as Gelfand, 1989, PCR Technology.Principles and Applications for DNA Amplification, H.A. Erlich, ed.,Stockton Press, New York; and Current Protocols In Molecular Biology,Vol. 2, Ch. 15, Ausubel et al., eds 1988, New York, Wiley & Sons, Inc.

[0079] A PCR amplified sequence may be molecularly cloned and sequenced.The amplified sequence may utilized as a probe to isolate genomic orcDNA clones of a SCR gene, as described below. This, in turn, willpermit the determination of a SCR gene′s complete nucleotide sequence,including its promoter, the analysis of its expression, and theproduction of its encoded protein, as described infra.

[0080] In a preferred embodiment, PCR amplification of SCR gene and/orcoding sequences can be carried out according to the followingprocedure:

[0081] Primers

[0082] Forward

[0083] Name: SCR5AII (23-mer, 2 inosines, 64-mix)

[0084] A.A. code: HFTANQAI

[0085] DNA Sequence: 5′ CAT/C TTT/C ACI GCI AAT/C CAA/G GCN AT 3′

[0086] Name: SCR5B (29-mer, 1 inosine, 144-mix)

[0087] A.A. code: VHIID(L/F)D

[0088] DNA Sequence: 5′ ACGTCTCGA GTI CAT/C ATA/C/T ATA/C/T GAT/C TTN GA3′

[0089] Name: 1F

[0090] A.A. code: LQCAEAV

[0091] DNA Sequence: (T/C)TI CA(A/G) TG(T/C GCI GA(A/G) GCN GT

[0092] Reverse:

[0093] Name: SCR3AII (23-mer, 2 inosines, 128-mix)

[0094] A.A. code: PGGPP(H/N/K)(V/L/F)R′

[0095] DNA Sequence: 5′ CG/T CCA/C GTG/T TGG IGG ICC NCC NGG 3′

[0096] Name: 1R

[0097] A.A. code: AFQVFNGI

[0098] DNA Sequence: AT ICC (A/G)TT (A/G)AA IAC (C/T)TG (A/G)AA NGC

[0099] Name: 4R

[0100] A.A. code: QWPGLFHI

[0101] DNA Sequence: AT (A/G)TG (A/G)AA IA(A/G) NCC IGG CCA (C/T)TG

[0102] I=inosine

[0103] N=A/C/G/T

[0104] Useful primer combinations include the following:

[0105] SCR5AII+SCR3AII; SCR5B+SCR3AII; IF+IR; and IF+4R

[0106] PCR

[0107] Reaction mixture (volume 50 μl):

[0108] 5 μl 10×amplification buffer containing Mg (Boehringer-Mannheim)

[0109] 1 μl 10 mM dNTP's

[0110] 1 μl forward primer (stock concentration: 80 pmol/μl)

[0111] 1 μl reverse primer (80 pmol/μl)

[0112] DNA (100-300 ng).

[0113] Begin reaction with “hot start” in which the enzyme is added tothe mix only after a brief denaturation at a high temperature (80° C.)

[0114] Cycles

[0115] 94° C. 30 sec—brief denaturation (to prevent non-specificpriming)

[0116] 80° C. 5 min—apply the enzyme to the tubes (30 tubes/round atmaximum)

[0117] 94° C. 5 min—thorough denaturation

[0118] 2 times:

[0119] 94° C. 1 min

[0120] 64° C. 5 min

[0121] 72° C. 2 min 2 times:

[0122] 940° C. 1 min

[0123] 62° C. 5 min

[0124] 72° C. 2 min 2 times:

[0125] 94° C. 1 min

[0126] 60° C. 5 min

[0127] 72° C. 2 min

[0128] (reduce the annealing temperature 20° C. in every second round),until 44° C. is reached after that:

[0129] 40 times:

[0130] 94° C. 20 sec

[0131] 48° C. 1 min

[0132] 72° C. 2 min

[0133] finally, let cool down to 15° C.

[0134] A SCR gene coding sequence may also be isolated by screening aplant genomic or cDNA library using a SCR nucleotide sequence (e.g., thesequence of any of the SCR genes and sequences and EST clone sequenceslisted in Table 1.) as hybridization probe. For example, the whole or asegment of the Arabidopsis SCR nucleotide sequence (FIG. 5A) may beused. Alternatively, a SCR gene may be isolated from such librariesusing as probe a degenerate oligonucleotide that corresponds to asegment of a SCR amino acid sequence. For example, degenerateoligonucleotide probe corresponding to a segment of the Arabidopsis SCRamino acid sequence (FIG. 5E) may be used.

[0135] In preparation of cDNA libraries, total RNA is isolated fromplant tissues, preferably roots. Poly(A)+ RNA is isolated from the totalRNA, and cDNA prepared from the poly(A)+ RNA, all using standardprocedures. See, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed., Vol. 2 (1989). The cDNAs may be synthesizedwith a restriction enzyme site at their 3′-ends by using an appropriateprimer and further have linkers or adaptors attached at their 5′-ends tofacilitate the insertion of the cDNAs into suitable CDNA cloningvectors. Alternatively, adaptors or linkers may be attached to the cDNAsafter the completion of cDNA synthesis.

[0136] In preparation of genomic libraries, plant DNA is isolated andfragments are generated, some of which will encode parts of the wholeSCR protein. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNase in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication. The DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis, column chromatographyand sucrose gradient centrifugation.

[0137] The genomic DNA or cDNA fragments can be inserted into suitablevectors, including but not limited to, plasmids, cosmids, bacteriophageslambda or T₄, and yeast artificial chromosome (YAC) [See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Glover,D. M (ed.), DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford,U.K., Vols. I and II (1985)].

[0138] The SCR nucleotide probe, DNA or RNA, should be at least 17nucleotides, preferably at least 26 nucleotides, and most preferably atleast 50 nucleotides in length. The nucleotide probe is hybridized undermoderate stringency conditions and washed under moderate, preferablyhigh stringency conditions. Clones in libraries with insert DNA havingsubstantial homology to the SCR probe will hybridize to the probe.Hybridization of the nucleotide probe to genomic or CDNA libraries iscarried out using methods known in the art. One of ordinary skill in theart will know that the appropriate hybridization and wash conditionsdepend on the length and base composition of the probe and that suchconditions may be determined using standard formulae. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Vol. 2, (1989)pp 11.45-11.57 and 15.55-15.57.

[0139] The identity of a cloned or amplified SCR gene sequence can beverified by comparing the amino acid sequences of its three open readingframes with the amino acid sequence of a SCR gene (e.g., Arabidopsis SCRprotein [SEQ ID No:2]). A SCR gene or coding sequence encodes a proteinor polypeptide whose amino acid sequence is substantially similar tothat of a SCR protein or polypeptide (e.g., the amino acid sequence ofany one of the SCR proteins and/or polypeptides shown in FIG. 5A, 5E,FIG. 8, FIG. 9, FIGS. 11A-B, FIGS. 15A-S, FIG. 17B and FIG. 18). Theidentity of the cloned or amplified SCR gene sequence may be furtherverified by examining its expression pattern, which should show highlylocalized expression in the embryo and/or root of the plant from whichthe SCR gene sequence was isolated.

[0140] Comparison of the amino acid sequences encoded by a cloned oramplified sequence may reveal that it does not contain the entire SCRgene or its promoter. In such a case the cloned or amplified SCR genesequence may be used as a probe to screen a genomic library for cloneshaving inserts that overlap the cloned or amplified SCR gene sequence. Acomplete SCR gene and its promoter may be reconstructed by splicing theoverlapping SCR gene sequences.

[0141] 5.1.2. Expression Of SCR Gene Products

[0142] SCR proteins, polypeptides and peptide fragments, mutated,truncated or deleted forms of SCR and/or SCR fusion proteins can beprepared for a variety of uses, including but not limited to thegeneration of antibodies, as reagents in assays, the identification ofother cellular gene products involved in regulation of root development;etc.

[0143] SCR translational products include, but are not limited to thoseproteins and polypeptides encoded by the SCR gene sequences described inSection 5.1, above. The invention encompasses proteins that arefunctionally equivalent to the SCR gene products described in Section5.1. Such a SCR gene product may contain one or more deletions,additions or substitutions of SCR amino acid residues within the aminoacid sequence encoded by any one of the SCR gene sequences described,above, in Section 5.1, but which result in a silent change, thusproducing a functionally equivalent SCR gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

[0144] For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent”, as utilized herein, refers toa protein capable of exhibiting a substantially similar in vivo activityas the endogenous SCR gene products encoded by the SCR gene sequencesdescribed in Section 5.1, above. Alternatively, “functionallyequivalent” may refer to peptides capable of regulating gene expressionin a manner substantially similar to the way in which the correspondingportion of the endogenous SCR gene product would.

[0145] The invention also encompasses mutant SCR proteins andpolypeptides that agree not functionally equivalent to the gene productsdescribed in Section 5.1. Such a mutant SCR protein or polypeptide maycontain one or more deletions, additions or substitutions of SCR aminoacid residues within the amino acid sequence encoded by any one the SCRgene sequences described above in Section 5.1., and which result in lossof one or more functions of the SCR protein (e.g., recognition of aspecific nucleic sequence, binding of an transcription factor, etc.),thus producing a SCR gene product not functionally equivalent to thewild-type SCR protein.

[0146] While random mutations can be made to SCR DNA (using randommutagenesis techniques well known to those skilled in the art) and theresulting mutant SCRs tested for activity, site-directed mutations ofthe SCR gene and/or coding sequence can be engineered (usingsite-directed mutagenesis techniques well known to those skilled in theart) to generate mutant SCRs with increased function, (e.g., resultingin improved root formation), or decreased function (e.g., resulting insuboptimal root function). In particular, mutated SCR proteins in whichany of the domains shown in FIGS. 13A-F are deleted or mutated arewithin the scope of the invention. Additionally, peptides correspondingto one or more domains of the SCR (e.g., shown in FIGS. 13A-F),truncated or deleted SCRs, as well as fusion proteins in which the fulllength SCR, a SCR polypeptide or peptide fused to an unrelated proteinare also within the scope of the invention and can be designed on thebasis of the SCR nucleotide and SCR amino acid sequences disclosed inSection 5.1. above.

[0147] While the SCR polypeptides and peptides can be chemicallysynthesized (e.g., see Creighton, 1983, Proteins: Structures andMolecular Principles, W. H. Freeman & Co., N.Y.) large polypeptidesderived from SCR and the full length SCR may advantageously be producedby recombinant DNA technology using techniques well known to thoseskilled in the art for expressing nucleic acid sequences.

[0148] Methods which are well known to those skilled in the art can beused to construct expression vectors containing SCR protein codingsequences and appropriate transcriptional/translational control signals.These methods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Sambrook et al., 1989,supra, and Ausubel et al., 1989, supra. Alternatively, RNA capable ofencoding SCR protein sequences may be chemically synthesized using, forexample, synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford.

[0149] A variety of host-expression vector systems may be utilized toexpress the SCR gene products of the invention. Such host-expressionsystems represent vehicles by which the SCR gene products of interestmay be produced and subsequently recovered and/or purified from theculture or plant (using purification methods well known to those skilledin the art), but also represent cells which may, when transformed ortransfected with the appropriate nucleotide coding sequences, exhibitthe SCR protein of the invention in situ. These include but are notlimited to microorganisms such as bacteria (e.g., E. coli, B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing SCR protein coding sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing the SCR protein coding sequences; insectcell systems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the SCR protein coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing SCR protein coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter; thecytomegalovirus promoter/enhancer; etc.).

[0150] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the SCRprotein being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of antibodies or to screenpeptide libraries, for example, vectors which direct the expression ofhigh levels of fusion protein products that are readily purified may bedesirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in whichthe SCR coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-10 3109;Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with gluta-thione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene protein can be released from the GST moiety.

[0151] In one such embodiment of a bacterial system, full length cDNAsequences are appended with in-frame Bam HI sites at the amino terminusand Eco RI sites at the carboxyl terminus using standard PCRmethodologies (Innis et al., 1990, supra) and ligated into the pGEX-2TKvector (Pharmacia, Uppsala, Sweden). The resulting cDNA constructcontains a kinase recognition site at the amino terminus for radioactivelabelling and glutathione S-transferase sequences at the carboxylterminus for affinity purification (Nilsson, et al., 1985, EMBO J. 4:1075; Zabeau and Stanley, 1982, EMBO J. 1: 1217.

[0152] The recombinant constructs of the present invention may include aselectable marker for propagation of the construct. For example, aconstruct to be propagated in bacteria preferably contains an antibioticresistance gene, such as one that confers resistance to kanamycin,tetracycline, streptomycin, or chloramphenicol. Suitable vectors forpropagating the construct include plasmids, cosmids, bacteriophages orviruses, to name but a few.

[0153] In addition, the recombinant constructs may includeplant-expressible, selectable, or screenable marker genes for isolating,identifying or tracking plant cells transformed by these constructs.Selectable markers include, but are not limited to, genes that conferantibiotic resistance, (e.g., resistance to kanamycin or hygromycin) orherbicide resistance (e.g., resistance to sulfonylurea,phosphinothricin, or glyphosate). Screenable markers include, but arenot be limited to, genes encoding β-glucuronidase (Jefferson, 1987,Plant Mol. Biol. Rep. 5:387-405), luciferase (Ow et al., 1986, Science234:856-859), B protein that regulates anthocyanin pigment production(Goff et al., 1990, EMBO J 9:2517-2522).

[0154] In embodiments of the present invention which utilize theAgrobacterium tumefacien system for transforming plants (see infra), therecombinant constructs may additionally comprise at least the rightT-DNA border sequences flanking the DNA sequences to be transformed intothe plant cell. Alternatively, the recombinant constructs may comprisethe right and left T-DNA border sequences flanking the DNA sequence. Theproper design and construction of such T-DNA based transformationvectors are well known to those skilled in the art.

[0155] 5.1.3. Antibodies To SCR Proteins And Polypeptides

[0156] Antibodies that specifically recognize one or more epitopes ofSCR, or epitopes of conserved variants of SCR, or peptide fragments ofthe SCR are also encompassed by the invention. Such antibodies includebut are not limited to polyclonal antibodies, monoclonal antibodies(mAbs), humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0157] For the production of antibodies, various host animals may beimmunized by injection with the SCR protein, an SCR peptide (e.g., onecorresponding to a functional domain of the protein), a truncated SCRpolypeptide (SCR in which one or more domains has been deleted),functional equivalents of the SCR protein, or mutants of the SCRprotein. Such SCR proteins, polypeptides, peptides or fusion proteinscan be prepared and obtained as described in Section 5.1.2. supra. Hostanimals may include but are not limited to rabbits, mice, and rats, toname but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

[0158] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (Nature 256:495-497[1975];and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan ,R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0159] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-5 454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

[0160] In addition, techniques have been developed for the production ofhumanized antibodies. (See, e.g., Queen, U.S. Pat. No. 5,585,089.) Animmunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, referredto as complementarily determining regions (CDRs). The extent of theframework region and CDRs have been precisely defined (see, “Sequencesof Proteins of Immunological Interest”, Kabat, E. et al., U.S.Department of Health and Human Services (1983). Briefly, humanizedantibodies are antibody molecules from non-human species having one ormore CDRs from the non-human species and a framework region from a humanimmunoglobulin molecule.

[0161] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against SCR proteins or polypeptides.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0162] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0163] Antibodies to a SCR protein and/or polypeptide can, in turn, beutilized to generate anti-idiotype antibodies that “mimic” SCR, usingtechniques well known to those skilled in the art. (See, e.g., Greenspan& Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438).

[0164] 5.1.4. SCR Gene Or Gene Products As Markers For Qualitative TraitLoci

[0165] Any of the nucleotide sequences (including EST clone sequences)described in §§5.1 and 5.1.1. and/or listed in Table 1, and/orpolypeptides and proteins described in §§ 5.1.2. and/or listed in Table1, can be used as markers for qualitative trait loci in breedingprograms for crop plants. To this end, the nucleic acid molecules,including but not limited to full length SCR coding sequences, and/orpartial sequences (ESTs), can be used in hybridization and/or DNAamplification assays to identify the endogenous SCR genes, scr mutantalleles and/or SCR expression products in cultivars as compared towild-type plants. They can also be used as markers for linkage analysisof qualitative trait loci. It is also possible that the SCR gene mayencode a product responsible for a qualitative trait that is desirablein a crop breeding program. Alternatively, the SCR protein, peptidesand/or antibodies can be used as reagents in immunoassays to detectexpression of the SCR gene in cultivars and wild-type plants.

[0166] 5.2. SCR Promoters

[0167] According to the present invention, SCR promoters and functionalportions thereof described herein refer to regions of the SCR gene whichare capable of promoting tissue-specific expression in embryos and/orroots of an operably linked coding sequence in plants. The SCR promoterdescribed herein refers to the regulatory elements of SCR genes, i.e.,regulatory regions of genes which are capable of selectively hybridizingto the nucleic acids described in Section 5.1, or regulatory sequencescontained, for example, in the region between the translational startsite of the Arabidopsis SCR gene and the HindIII site approximately 2.5kb upstream of the site in plasmid pLIG1-3/SAC+Mob21SAC (see FIGS. 5Aand 14) in hybridization assays, or which are homologous by sequenceanalysis (containing a span of 10 or more nucleotides in which at least50 percent of the nucleotides are identical to the sequences presentedherein). Homologous nucleotide sequences refer to nucleotide sequencesincluding, but not limited to, SCR promoters in diverse plant species(e.g., promoters of orthologs of Arabidopsis SCR) as well as geneticallyengineered derivatives of the promoters described herein.

[0168] Methods which could be used for the synthesis, isolation,molecular cloning, characterization and manipulation of SCR promotersequences are well known to those skilled in the art. See, e.g., thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd. ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989).

[0169] According to the present invention, SCR promoter sequences orportions thereof described herein may be obtained from appropriate plantor mammalian sources from cell lines or recombinant DNA constructscontaining SCR promoter sequences, and/or by chemical synthetic methods.SCR promoter sequences can be obtained from genomic clones containingsequences 5′ upstream of SCR coding sequences. Such 5′ upstream clonesmay be obtained by screening genomic libraries using SCR protein codingsequences, particularly those encoding SCR N-terminal sequences, fromSCR gene clones obtained as described in Sections 5.1. and 5.2. Standardmethods that may used in such screening include, for example, the methodset forth in Benton & Davis, 1977, Science 196:180 for bacteriophagelibraries; and Grunstein & Hogness, 1975, Proc. Nat. Acad. Sci. U.S.A.72:3961-3965 for plasmid libraries.

[0170] The full extent and location of SCR promoters within such 5′upstream clones may be determined by the functional assay describedbelow. In the event a 5′ upstream clone does not contain the entire SCRpromoter as determined by the functional assay, the insert DNA of theclone may be used to isolate genomic clones containing sequences further5′ upstream of the SCR coding sequences. Such further upstream sequencescan be spliced on to existing 5′ upstream sequences and thereconstructed 5′ upstream region tested for functionality as a SCRpromoter (i.e., promoting tissue-specific expression in embryos and/orroots of an operably linked gene in plants). This process may be repeatuntil the complete SCR promoter is obtained.

[0171] The location of the SCR promoter within genomic sequences 5′upstream of the SCR gene isolated as described above may be determinedusing any method known in the art. For example, the 3′-end of thepromoter may be identified by locating the transcription initiationsite, which may be determined by methods such as RNase protection (e.g.,Liang et al., 1989, J. Biol. Chem. 264:14486-14498), primer extension(e.g., Weissenborn & Larson, 1992, J. Biol. Chem. 267:6122-6131), and/orreverse transcriptase/PCR. The location of the 3′-end of the promotermay be confirmed by sequencing and computer analysis, examining for thecanonical AGGA or TATA boxes of promoters that are typically 50-60 basepairs (bp) and 25-35 bp 5′-upstream of the transcription initiationsite. The 5′-end promoter may be defined by deleting sequences from the5′-end of the promoter containing fragment, constructing atranscriptional or translational fusion of the resected fragment and areporter gene, and examining the expression characteristics of thechimeric gene in transgenic plants. Reporter genes that may be used tosuch ends include, but are not limited to, GUS, CAT, luciferase,β-galactosidase and C1 and R gene controlling anthocyanin production.

[0172] According to the present invention, a SCR promoter is one thatconfers to an operably linked gene in a transgenic plant tissue-specificexpression in roots, root nodules, stems and/or embryos. A SCR promotercomprises the region between about −5,000 bp and +1 bp upstream of thetranscription initiation site of SCR gene. In a particular embodiment,the Arabidopsis SCR promoter comprises the region between positions −2.5kb and +1 in the 5′ upstream region of the Arabidopsis SCR gene (seeFIGS. 5A and 14).

[0173] 5.2.1. CIS-Regulatory Elements Of SCR Promoters

[0174] According to the present invention, the cis-regulatory elementswithin a SCR promoter may be identified using any method known in theart. For example, the location of cis-regulatory elements within aninducible promoter may be identified using methods such as DNase orchemical footprinting (e.g., Meier et al., 1991, Plant Cell 3:309-315)or gel retardation (e.g., Weissenborn & Larson, 1992, J. Biol. Chem.267-6122-6131; Beato, 1989, Cell 56:335-344; Johnson et al., 1989, Ann.Rev. Biochem. 58:799-839). Additionally, resectioning experiments mayalso be employed to define the location of the cis-regulatory elements.For example, an inducible promoter-containing fragment may be resectedfrom either the 5′ or 3′-end using restriction enzyme or exonucleasedigests.

[0175] To determine the location of cis-regulatory elements within thesequence containing the inducible promoter, the 5′- or 3′-resectedfragments, internal fragments to the inducible promoter containingsequence, or inducible promoter fragments containing sequencesidentified by footprinting or gel retardation experiments may be fusedto the 5′-end of a truncated plant promoter, and the activity of thechimeric promoter in transgenic plant examined. Useful truncatedpromoters to these ends comprise sequences starting at or about thetranscription initiation site and extending to no more than 150 bp 5′upstream. These truncated promoters generally are inactive or are onlyminimally active. Examples of such truncated plant promoters mayinclude, among others, a “minimal” CaMV 35S promoter whose 5′ endterminates at position -46 bp with respect to the transcriptioninitiation site (Skriver et al., Proc. Natl. Acad. Sci. USA88:7266-7270); the truncated “-90 35S” promoter in the X-GUS-90 vector(Benfey & Chua, 1989, Science 244:174-181); a truncated “-101 nos”promoter derived from the nopaline synthase promoter (Aryan et al.,1991, Mol. Gen. Genet. 225:65-71); and the truncated maize Adh-1promoter in pADcat 2 (Ellis et al., 1987, EMBO J. 6:11-16).

[0176] According to the present invention, a cis-regulatory element of aSCR promoter is a sequence that confers to a truncated promotertissue-specific expression in embryos, stems, root nodules and/or roots.

[0177] 5.2.2. SCR PROMOTER-DRIVEN EXPRESSION VECTORS

[0178] The properties of the nucleic acid sequences are varied as arethe genetic structures of various potential host plant cells. In thepreferred embodiments of the present invention, described herein, anumber of features which an artisan may recognize as not beingabsolutely essential, but clearly advantageous are used. These includemethods of isolation, synthesis or construction of gene constructs, themanipulation of the gene constructs to be introduced into plant cells,certain features of the gene constructs, and certain features of thevectors associated with the gene constructs.

[0179] Further, the gene constructs of the present invention may beencoded on DNA or RNA molecules. According to the present invention, itis preferred that the desired, stable genotypic change of the targetplant be effected through genomic integration of exogenously introducednucleic acid construct(s), particularly recombinant DNA constructs.Nonetheless, according to the present invention, such genotypic changescan also be effected by the introduction of episomes (DNA or RNA) thatcan replicate autonomously and that are somatically and germinallystable. Where the introduced nucleic acid constructs comprise RNA, planttransformation or gene expression from such constructs may proceedthrough a DNA intermediate produced by reverse transcription.

[0180] The present invention provides for use of recombinant DNAconstructs which contain tissue-specific and developmental-specificpromoter fragments and functional portions thereof. As used herein, afunctional portion of a SCR promoter is capable of functioning as atissue-specific promoter in the embryo, stem, root nodule and/or root ofa plant. The functionality of such sequences can be readily establishedby any method known in the art. Such methods include, for example,constructing expression vectors with such sequences and determiningwhether they confer tissue-specific expression in the embryo, stem, rootnodule and/or root to an operably linked gene. In a particularembodiment, the invention provides for the use of the Arabidopsis SCRpromoter contained in the sequences depicted in FIGS. 5A and 14 and theinsert DNA of plasmid pGEX-2TK⁺.

[0181] The SCR promoters of the invention may be used to direct theexpression of any desired protein, or to direct the expression of a RNAproduct, including, but not limited to, an “antisense” RNA or ribozyme.Such recombinant constructs generally comprise a native SCR promoter ora recombinant SCR promoter derived therefrom, ligated to the nucleicacid sequence encoding a desired heterologous gene product.

[0182] A recombinant SCR promoter is used herein to refer to a promoterthat comprises a functional portion of a native SCR promoter or apromoter that contains native promoter sequences that is modified by aregulatory element from a SCR promoter. Alternatively, a recombinantinducible promoter derived from the scr promoter may be a chimericpromoter, comprising a full-length or truncated plant promoter modifiedby the attachment of one or more SCR cis-regulatory elements.

[0183] The manner of chimeric promoter constructions may be any wellknown in the art. For examples of approaches that can be used in suchconstructions, see Section 5.1.2., above and Fluhr et al., 1986, Science232:1106-1112; Ellis et al., 1987, EMBO J. 6:11-16; Strittmatter & Chua,1987, Proc. Natl. Acad. Sci. USA 84:8986-8990; Poulsen & Chua, 1988,Mol. Gen. Genet. 214:16-23; Comai et al., 1991, Plant Mol. Biol.15:373-381; Aryan et al., 1991, Mol. Gen. Genet. 225:65-71.

[0184] According to the present invention, where a SCR promoter or arecombinant SCR promoter is used to express a desired protein, the DNAconstruct is designed so that the protein coding sequence is ligated inphase with the translational initiation codon downstream of thepromoter. Where the promoter fragment is missing 5′ leader sequences, aDNA fragment encoding both the protein and its 5′ RNA leader sequence isligated immediately downstream of the transcription initiation site.Alternatively, an unrelated 5′ RNA leader sequence may be used to bridgethe promoter and the protein coding sequence. In such instances, thedesign should be such that the protein coding sequence is ligated inphase with the initiation codon present in the leader sequence, orligated such that no initiation codon is interposed between thetranscription initiation site and the first methionine codon of theprotein.

[0185] Further, it may be desirable to include additional DNA sequencesin the protein expression constructs. Examples of additional DNAsequences include, but are not limited to, those encoding: a 3′untranslated region; a transcription termination and polyadenylationsignal; an intron; a signal peptide (which facilitates the secretion ofthe protein); or a transit peptide (which targets the protein to aparticular cellular compartment such as the nucleus, chloroplast,mitochondria, or vacuole).

[0186] 5.3. Production of Transgenic Plants and Plant Cells

[0187] According to the present invention, a desirable plant or plantcell may be obtained by transforming a plant cell with the nucleic acidconstructs described herein. In some instances, it may be desirable toengineer a plant or plant cell with several different gene constructs.Such engineering may be accomplished by transforming a plant or plantcell with all of the desired gene constructs simultaneously.Alternatively, the engineering may be carried out sequentially. That is,transforming with one gene construct, obtaining the desired transformantafter selection and screening, transforming the transformant with asecond gene construct, and so on.

[0188] In an embodiment of the present invention, grobacterium isemployed to introduce the gene constructs into plants. Suchtransformations preferably use binary grobacterium T-DNA vectors (Bevan,1984, Nuc. Acid Res. 12:8711-8721), and the co-cultivation procedure(Horsch et al., 1985, Science 227:1229-1231). Generally, theAgrobacterium transformation system is used to engineer dicotyledonousplants (Bevan et al., 1982, Ann. Rev. Genet. 16:357-384; Rogers et al.,1986, Methods Enzymol. 118:627-641). The Agrobacterium transformationsystem may also be used to transform, as well as transfer, DNA tomonocotyledonous plants and plant cells (see Hernalsteen et al., 1984,EMBO J 3:3039-3041; Hooykass-Van Slogteren et al., 1984, Nature311:763-764; Grimsley et al., 1987, Nature 325:1677-179; Boulton et al.,1989, Plant Mol. Biol. 12:31-40.; Gould et al., 1991, Plant Physiol.95:426-434).

[0189] In other embodiments, various alternative methods for introducingrecombinant nucleic acid constructs into plants and plant cells may alsobe utilized. These other methods are particularly useful where thetarget is a monocotyledonous plant or plant cell. Alternative genetransfer and transformation methods include, but are not limited to,protoplast transformation through calcium-, polyethylene glycol (PEG)-or electroporation-mediated uptake of naked DNA (see Paszkowski et al.,1984, EMBO J 3:2717-2722, Potrykus et al., 1985, Mol. Gen. Genet.199:169-177; Fromm et al., 1985, Proc. Natl. Acad. Sci. USA82:5824-5828; Shimamoto, 1989, Nature 338:274-276), and electroporationof plant tissues (D′Halluin et al., 1992, Plant Cell 4:1495-1505).Additional methods for plant cell transformation include microinjection,silicon carbide mediated DNA uptake (Kaeppler et al., 1990, Plant CellReporter 9:415-418), and microprojectile bombardment (see Klein et al.,1988, Proc. Natl. Acad. Sci. USA 85:4305-4309; Gordon-Kamm et al., 1990,Plant Cell 2:603-618).

[0190] According to the present invention, a wide variety of plants maybe engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe instant invention and the various transformation methods mentionedabove. In preferred embodiments, target plants for engineering include,but are not limited to, crop plants such as maize, wheat, rice, soybean,tomato, tobacco, carrots, peanut, potato, sugar beets, sunflower, yam,Arabidopsis, rape seed, and petunia; and trees such as spruce.

[0191] According to the present invention, desired plants and plantcells may be obtained by engineering the gene constructs describedherein into a variety of plant cell types, including but not limited to,protoplasts, tissue culture cells, tissue and organ explants, pollen,embryos as well as whole plants. In an embodiment of the presentinvention, the engineered plant material is selected or screened fortransformants (i.e., those that have incorporated or integrated theintroduced gene construct(s)) following the approaches and methodsdescribed below. An isolated transformant may then be regenerated into aplant. Alternatively, the engineered plant material may be regeneratedinto a plant, or plantlet, before subjecting the derived plant, orplantlet, to selection or screening for the marker gene traits.Procedures for regenerating plants from plant cells, tissues or organs,either before or after selecting or screening for marker gene(s), arewell known to those skilled in the art.

[0192] A transformed plant cell, callus, tissue or plant may beidentified and isolated by selecting or screening the engineered plantmaterial for traits encoded by the marker genes present on thetransforming DNA. For instance, selection may be performed by growingthe engineered plant material on media containing inhibitory amounts ofthe antibiotic or herbicide to which the transforming marker geneconstruct confers resistance. Further, transformed plants and plantcells may also be identified by screening for the activities of anyvisible marker genes (e.g., the β-glucuronidase, luciferase, B or C1genes) that may be present on the recombinant nucleic acid constructs ofthe present invention. Such selection and screening methodologies arewell known to those skilled in the art.

[0193] Physical and biochemical methods may also be used to identify aplant or plant cell transformant containing the gene constructs of thepresent invention. These methods include but are not limited to: 1)Southern analysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert; 2) Northern blot, S-1 RNaseprotection, primer-extension or reverse transcriptase-PCR amplificationfor detecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins; 5) biochemical measurements of compounds produced as aconsequence of the expression of the introduced gene constructs.Additional techniques, such as in situ hybridization, enzyme staining,and immunostaining, may also be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the art.

[0194] 5.3.1. Transgenic Plants That Ectopically Express SCR

[0195] In accordance to the present invention, a plant that expresses arecombinant SCR gene may be engineered by transforming a plant cell witha gene construct comprising a plant promoter operably associated with asequence encoding SCR protein or a fragment thereof. (Operablyassociated is used herein to mean that transcription controlled by the“associated” promoter would produce a functional messenger RNA, whosetranslation would produce the enzyme.) The plant promoter may beconstitutive or inducible. Useful constitutive promoters include, butare not limited to, the CaMV 35S promoter, the T-DNA mannopinesynthetase promoter, and their various derivatives. Useful induciblepromoters include but are not limited to the promoters of ribulosebisphosphate carboxylase (RUBISCO) genes, chlorophyll a/b bindingprotein (CAB) genes, heat shock genes, the defense responsive gene(e.g., phenylalanine ammonia lyase genes), wound induced genes (e.g.,hydroxyproline rich cell wall protein genes), chemically-inducible genes(e.g., nitrate reductase genes, gluconase genes, chitinase genes, PR-1genes etc.), dark-inducible genes (e.g., asparagine synthetase gene(Coruzzi and Tsai, U.S. Pat. No. 5,256,558, Oct. 26, 1993, Gene EncodingPlant Asparagine Synthetase) developmentally regulated genes (e.g.,Shoot Meristemless gene) to name just a few.

[0196] In yet another embodiment of the present invention, it may beadvantageous to transform a plant with a gene construct operably linkinga modified or artificial promoter to a sequence encoding SCR protein ora fragment thereof. Typically, such promoters, constructed byrecombining structural elements of different promoters, have uniqueexpression patterns and/or levels not found in natural promoters. See,e.g., Salina et al., 1992, Plant Cell 4:1485-1493, for examples ofartificial promoters constructed from combining cis-regulatory elementswith a promoter core.

[0197] In a preferred embodiment of the present invention, theassociated promoter is a strong and root, root nodule, stem and/orembryo-specific plant promoter such that the SCR protein isoverexpressed in the transgenic plant. Examples of root- and rootnodules-specific promoters include but are not limited to the promotersof SCR genes, SHR genes, legehemoglobin genes, nodulin genes androot-specific glutamine synthetase genes (See e.g., Tingey et al., 1987,EMBO J. 6:1-9; Edwards et al., 1990, Proc. Nat. Acad. Sci. USA87:3459-3463).

[0198] In yet another preferred embodiment of the present invention, theoverexpression of SCR protein in roots may be engineered by increasingthe copy number of the SCR gene. One approach to producing suchtransgenic plants is to transform with nucleic acid constructs thatcontain multiple copies of the complete SCR gene (i.e., with its ownnative scr promoter). Another approach is repeatedly transformsuccessive generations of a plant line with one or more copies of thecomplete SCR gene. Yet another approach is to place a complete SCR genein a nucleic acid construct containing an amplification-selectablemarker (ASM) gene such as the glutamine synthetase or dihydrofolatereductase gene. Cells transformed with such constructs is subjected toculturing regimes that select cell lines with increased copies ofcomplete SCR gene. See, e.g., Donn et al., 1984, J. Mol. Appl. Genet.2:549-562, for a selection′ protocol used to isolate of a plant cellline containing amplified copies of the GS gene. Because the desiredgene is closely linked to the ASM, cell lines that amplified the ASMgene are also likely to have amplified the SCR gene. Cell lines withamplified copies of the SCR gene can then be regenerated into transgenicplants.

[0199] 5.3.2. Transgenic Plants That Suppress Endogenous SCR Expression

[0200] In accordance with the present invention, a desired plant may beengineered by suppressing SCR activity. In one embodiment, thesuppression may be engineered by transforming a plant with a geneconstruct encoding an antisense RNA or ribozyme complementary to asegment or the whole of SCR RNA transcript, including the mature targetmRNA. In another embodiment, SCR gene suppression may be engineered bytransforming a plant cell with a gene construct encoding a ribozyme thatcleaves the SCR mRNA transcript. Alternatively, the plant can beengineered, e.g., via targeted homologous recombination to inactive or“knock-out” expression of the plant's endogenous SCR.

[0201] For all of the aforementioned suppression constructs, it ispreferred that such gene constructs express specifically in the root,root nodule, stem and/or embryo tissues. Alternatively, it may bepreferred to have the suppression constructs expressed constitutively.Thus, constitutive promoters, such as the nopaline, CaMV 35S promoter,may also be used to express the suppression constructs. A most preferredpromoter for these suppression constructs is a SCR or SHR promoter.

[0202] In accordance with the present invention, desired plants withsuppressed target gene expression may also be engineered by transforminga plant cell with a co-suppression construct. A co-suppression constructcomprises a functional promoter operatively associated with a completeor partial SCR gene sequence. It is preferred that the operativelyassociated promoter be a strong, constitutive promoter, such as the CaMV35S promoter. Alternatively, the co-suppression construct promoter canbe one that expresses with the same tissue and developmental specificityas the scr gene.

[0203] According to the present invention, it is preferred that theco-suppression construct encodes a incomplete SCR mRNA, although aconstruct encoding a fully functional SCR mRNA or enzyme may also beuseful in effecting co-suppression.

[0204] In accordance with the present invention, desired plants withsuppressed target gene expression may also be engineered by transforminga plant cell with a construct that can effect site-directed mutagenesisof the SCR gene. (See, e.g., Offringa et al., 1990, EMBO J. 9:3077-84;and Kanevskii et al., 1990, Dokl. Akad. Nauk. SSSR 312:1505-1507) fordiscussions of nucleic constructs for effecting site-directedmutagenesis of target genes in plants.) It is preferred that suchconstructs effect suppression of SCR gene by replacing the endogenousSCR gene sequence through homologous recombination with none or inactiveSCR protein coding sequence.

[0205] 5.3.3. Transgenic Plants That Express A Transgene Controlled ByThe SCR Promoter SCR

[0206] In accordance with the present invention, a desired plant may beengineered to express a gene of interest under the control of the SCRpromoter. SCR promoters and functional portions thereof refer to regionsof the nucleic acid sequence which are capable of promotingtissue-specific transcription of an operably linked gene of interest inthe embryo, stem, root nodule and/or root of a plant. The SCR promoterdescribed herein refers to the regulatory elements of SCR genes asdescribed in Section 5.2.

[0207] Genes that may be beneficially expressed in the roots and/or rootnodules of plants include genes involved in nitrogen fixation orcytokines or auxins, or genes which regulate growth, or growth of roots.In addition, genes encoding proteins that confer on plants herbicide,salt, or pest resistance may be engineered for root specific expression.The nutritional value of root crops may also be enhanced through SCRpromoter driven expression of nutritional proteins. Alternatively,therapeutically useful proteins may be expressed specifically in rootcrops.

[0208] Genes that may be beneficially expressed in the stems of plantsinclude those involved in starch lignin or cellulose biosynthesis.

[0209] In accordance with the present invention, desired plants whichexpress a heterologous gene of interest under the control of the SCRpromoter may be engineered by transforming a plant cell with SCRpromoter driven constructs using those techniques described in Section5.2.2. and 5.3., supra.

[0210] 5.3.4. Screening Of Transfromed Plants For Those Having DesiredAltered Traits

[0211] It will be recognized by those skilled in the art that in orderto obtain transgenic plants having the desired engineered traits,screening of transformed plants (i.e., those having an gene construct ofthe invention) having those traits may be required. For example, wherethe plants have been engineered for ectopic overexpression of SCR gene,transformed plants are examined for those expressing the SCR gene at thedesired level and in the desired tissues and developmental stages. Wherethe plants have been engineered for suppression of the SCR gene product,transformed plants are examined for those expressing the SCR geneproduct (e.g., RNA or protein) at reduced levels in various tissues. Theplants exhibiting the desired physiological changes, e.g., ectopic SCRoverexpression or SCR suppression, may then be subsequently screened forthose plants that have the desired structural changes at the plant level(e.g., transgenic plants with overexpression or suppression of SCR genehaving the desired altered root structure). The same principle appliesto obtaining transgenic plants having tissue-specific expression of aheterologous gene in embryos and/or roots by the use of a SCR promoterdriven expression construct.

[0212] Alternatively, the transformed plants may be directly screenedfor those exhibiting the desired structural and functional changes. Inone embodiment, such screening may be for the size, length or pattern ofthe root of the transformed plants. In another embodiment, the screeningof the transformed plants may be for altered gravitropism or decreasedsusceptibility to lodging. In other embodiments, the screening of thetransformed plants may be for improved agronomic characteristics (e.g.,faster growth, greater vegetative or reproductive yields, or improvedprotein contents, etc.), as compared to unengineered progenitor plants,when cultivated under various growth conditions (e.g., soils or mediacontaining different amount of nutrients, water content).

[0213] According to the present invention, plants engineered with SCRoverexpression may exhibit improved vigorous growth characteristics whencultivated under conditions where large and thicker roots areadvantageous. Plants engineered for SCR suppression may exhibit improvedvigorous growth characteristics when cultivated under conditions wherethinner roots are advantageous.

[0214] Engineered plants and plant lines possessing such improvedagronomic characteristics may be identified by examining any offollowing parameters: 1) the rate of growth, measured in terms of rateof increase in fresh or dry weight; 2) vegetative yield of the matureplant, in terms of fresh or dry weight; 3) the seed or fruit yield; 4)the seed or fruit weight; 5) the total nitrogen content of the plant; 6)the total nitrogen content of the fruit or seed; 7) the free amino acidcontent of the plant; 8) the free amino acid content of the fruit orseed; 9) the total protein content of the plant; and 10) the totalprotein content of the fruit or seed. The procedures and methods forexamining these parameters are well known to those skilled in the art.

[0215] According to the present invention, a desired plant is one thatexhibits improvement over the control plant (i.e., progenitor plant) inone or more of the aforementioned parameters. In an embodiment, adesired plant is one that shows at least 5% increase over the controlplant in at least one parameter. In a preferred embodiment, a desiredplant is one that shows at least 20% increase over the control plant inat least one parameter. Most preferred is a plant that shows at least50% increase in at least one parameter.

6. EXAMPLE 1

[0216] Arabidopsis Scr Gene

[0217] This example describes the cloning and structure of theArabidopsis SCR gene and its expression. The deduced amino acid sequenceof the Arabidopsis SCR gene product contains a number of potentialfunctional domains similar to those found in transcription factors.Closely related sequences have been found in both dicots and monocotsindicating that Arabidopsis SCR is a member of a new protein family. Theexpression pattern of the SCR gene was characterized by means of in situhybridization and by an enhancer trap insertion upstream of the SCR gene(described in more detail in Section 7). The expression pattern isconsistent with a key role for Arabidopsis SCR in regulating theasymmetric division of the cortex/endodermis initial which is essentialfor generating the radial organization of the root.

[0218] 6.1. Materials and Methods

[0219] 6.1.1. Plant Culture

[0220] Arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col), andLandsberg erecta (Ler) were obtained from Lehle. Arabidopsis seeds weresurface sterilized and grown as described previously (Benfey et al.,1993, Development 119:57-70). Generation of the enhancer trap lines isdescribed in Section 7.

[0221] 6.1.2. Genetic Analysis

[0222] For the scr-1 allele, co-segregation of the mutant phenotype andkanamycin resistance conferred by the inserted T-DNA was determined asdescribed previously (Aeschbacher et al., 1995, Genes & Development9:330-340). Because kanamycin affects root growth, 1557 seeds fromheterozygous lines were germinated on non-selective media, scored forthe appearance of the mutant phenotype, and subsequently transferred toselective media. All (284) phenotypically mutant seedlings showedresistance to the antibiotic, whereas 834 of 1273 phenotypicallywild-type seedlings showed resistance to kanamycin, respectively.Phenotypically wild type plants (83) were also transferred to soil andallowed to set seeds. The progeny of these plants were plated onselective and non-selective media, and scored for the co-segregation ofthe mutant phenotype and antibiotic resistance. A majority (48) of theplants segregated for the mutant phenotype and for kanamycin resistance,whereas 35 were wild-type and sensitive to kanamycin. Due to amis-identified cross, scr-2 was originally thought to be non-allelic andwas named pinocchio (Scheres et al., 1995, Development 121:53-62).Subsequent mapping results placed it in an identical chromosomallocation as scr-1. The original scr-2 line contained at least two T-DNAinserts. Co-segregation analysis revealed a lack of linkage between theantibiotic resistance marker carried by the T-DNA and the mutantphenotype. Antibiotic sensitive lines were identified that segregatedfor mutants. These lines were crossed to scr-1. All F1 antibioticresistant progeny exhibited a mutant phenotype. All F2 progeny (fromindependent lines) were mutant, and there was a 3:1 segregation forantibiotic resistance indicating that the two mutations were allelic.Antibiotic sensitive lines of scr-2 were found to contain a rearrangedT-DNA insert as determined by Southern blots and PCR using T-DNAspecific probes and primers respectively. The presence of this T-DNA inthe SCR gene was confirmed by Southern blots using SCR probes. Acombination of T-DNA and SCR specific primers was used to amplifyT-DNA/SCR junctions. The PCR fragments were cloned using the TA cloningkit (Invitrogen) and sequenced. The insertion points were determined forboth 5′ and 3′ T-DNA/SCR junctions.

[0223] 6.1.3. Mapping

[0224] Mutant plants of scr-2 (WS background) were crossed to Col WT.DNA from mutant F2 individual plants were analyzed for co-segregationwith microsatellite (Bell & Ecker, 1994, Genomics 18:137-144) and CAPSmarkers (Konieczny & Ausubel, 1993, Plant J. 4:403-410). The closestlinkage was found to two CAPS markers located at the bottom ofchromosome III. Only one out of 238 mutant chromosomes was recombinantfor the BGL1 marker (Konieczny & Ausubel, 1993, Plant J. 4:403-410) andone out of 210 chromosomes was recombinant for the cdc2b marker.

[0225] A RFLP for the SCR gene was identified between Col and Lerecotypes with Xho I endonuclease. Genomic DNAs from independent R1 lines(Jarvis et al., 1994, Plant Mol. Biol. 24:685-687) were digested withXho I and blots were hybridized to SCR. Using the segregation dataobtained for 25 R1 lines, the SCR gene was mapped relative to molecularmarkers by CLUSTER. The SCR gene was assigned to the bottom ofchromosome III closest to BGL1.

[0226] 6.1.4. Phenotypic Analysis

[0227] Morphological characterization of the mutant roots was performedas follows: 7 to 14 days post-germination phenotypically mutantseedlings were fixed in 4.0% formaldehyde in PIPES buffer pH 7.2. Afterfixation the samples were dehydrated in ethanol followed by infiltrationwith Historesin (Jung-Leica, Heidelberg, Germany). Plastic sections weremounted on superfrost slides (Fisher). The sections were either stainedwith 0.05% toluidine blue and photographed using Kodak 160T film or usedfor Casparian strip detection or antibody staining.

[0228] Casparian strip detection was performed as described previously(Scheres et al., 1995, Development 121:53-62), with the followingmodifications. Plastic sections were used and the counterstaining wasdone in 0.1% aniline blue for 5 to 15 min. The sections were visualizedwith a Leitz fluorescent microscope with FITC filter. Pictures weretaken using a Leitz camera attached to the microscope and Kodak HC400film. Slides were digitized with a Nikon slide scanner and manipulatedin Adobe Photoshop.

[0229] For antibody staining, sections were blocked for 2 hours at roomtemperature in 1% BSA in PBS containing 0.1% Tween 20 (PBT). Sampleswere incubated with primary antibodies at 40° C. in 1% BSA in PBTovernight, and then washed 3 times 5 minutes each with PBT. Samples wereincubated for two hours with biotinylated secondary antibodies (VectorLaboratories) in PBT, and washed as above. Samples were incubated withTexas Red conjugated avidin D for 2 hours at room temperature, washed asbefore, and mounted in Citifluor. Immunofluorescence was observed with afluorescent microscope equipped with a Rhodamine filter. Staining withthe CCRC antibodies was performed as described previously (Freshour etal., 1996, Plant Physiol. 110:1413-1429).

[0230] 6.1.5. Molecular Techniques

[0231] Genomic DNA preparation was performed using the Elu-Quik kit(Schleicher & Schuell) protocol. Radioactive and non-radioactive DNAprobes were labeled with either random primed labeling or PCR-mediatedsynthesis according to the Genius kit manual (Boehringer Mannheim). E.coli and Agrobacterium tumefaciens cells were transformed using aBIO-RAD gene pulser. Plasmid DNA was purified using the alkaline lysismethod (Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.:Cold Spring Harbor Laboratory, 1982).

[0232] A probe made from a rescued fragment of 1.2 kb was used to screena wild-type genomic library made from WS plants. One genomic clonecontaining an insert of approximately 23 kb was isolated. A 3.0 kb Sac Ifragment from the genomic clone, which hybridized to the 1.2 kb probe,was subcloned and sequenced (FIG. 5A). Comparison of the nucleotidesequence between the genomic clone and the rescued plasmid revealed thesite of the T-DNA insertion. Approximately 600,000 plaques from a cDNAlibrary, obtained from inflorescences and siliques (Col ecotype), andtherefore enriched in embryos, were screened with the 1.2 kb probe. FourcDNA clones were isolated. The dideoxy sequencing method was performedusing the Sequenase kit (United States Biochemical Corp.).Sequence-specific internal primers were synthesized and used to sequencethe Sac I genomic as well the cDNA clones. Total RNA from plant tissueswas obtained using phenol/chloroform extractions as described in (Berryet al., 1985, Mol. Cell. Biol. 5:2238-2246) with minor modifications.Northern hybridization and detection were performed according to theGenius kit manual (Boehringer Mannheim).

[0233] To identify the site of insertion of the enhancer-trap T-DNA,genomic DNA from ET199 homozygous plants was amplified using primersspecific for the T-DNA left border and the SCR gene. An approximately2.0 kb fragment was amplified. This fragment was sequenced and the siteof insertion was found to be approximately 1 kb from the ATG startcodon.

[0234] 6.1.6. In Situ Hybridiztion

[0235] Antisense and sense SCR riboprobes were labeled withdigoxigenin-11-UTP (Boehringer Mannheim) using T7 polymerase followingthe manufacturer′s protocol. Probes contained a 1.1 kb 3′ portion of thecDNA. Probe purification, hydrolysis and quantification were performedas described in the Boehringer Mannheim Genius System user′s guide.

[0236] Tissue samples were fixed in 4% formaldehyde overnight at 4° C.and rinsed two times in PBS (Jackson et al., 1991, P1. Cell 3:115-125).They were subsequently pre-embedded in 1% agarose in PBS. The fixedtissue was dehydrated in ethanol, cleared in Hemo-De (Fisher Scientific,Pittsburgh, Pa.) and embedded in ParaplastPlus (Fisher Scientific).Tissue sections (10 μm thick) were mounted on SuperfrostPlus slides(Fisher Scientific). Section pretreatment and hybridization wereperformed according to (Lincoln et al., 1994, Plant Cell 6:1859-1876)except that proteinase K was used at mg/ml and a two hourprehybridization step was included. Probe concentration of 50 ng/ml/kbwas used in the hybridization.

[0237] Slides were washed and the immunological detection was performedaccording to (Coen et al., 1990, Cell 63:1311-1322) with the followingmodifications. Slides were first washed 5 h in 5×SSC, 50% formamide.After RNase treatment slides were rinsed three times (20 min each) inthe buffer (0.5 M NaCl, 10 mM Tris-HCl pH 8.0, 5.0 mM EDTA). In theimmunological detection, antibody was diluted 1:1000, levamisole (240ng/ml) was included in the detection buffer, and after stopping thereaction in 10 mM Tris, 1 mM EDTA, sections were mounted directly toAqua-Poly/Mount (Polysciences, Warrington, Pa.,).

[0238] 6.2. Results

[0239] 6.2.1. Characterization Of The SCR Phenotype

[0240] The scarecrow mutant scr-1 was isolated in a screen of T-DNAtransformed Arabidopsis lines (Feldmann, K.A., 1991, Plant J. 1:71-82),as a seedling with greatly reduced root length compared to wild-type(Scheres et al., 1995, Development 121:53-62). A second mutant scr-2with a similar phenotype was subsequently identified among T-DNAtransformed lines. Analysis of co-segregation between the mutantphenotype and antibiotic resistance carried by the T-DNA indicated tightlinkage for scr-1 and no linkage for scr-2 (see ExperimentalProcedures). An antibiotic sensitive line of scr-2 was isolated andcrossed with scr-1. The F2 progeny of this cross were all mutant andsegregated 3:1 for antibiotic resistance confirming allelism (seeMaterials & Methods). The principal phenotypic difference between thetwo alleles was that scr-1 root growth was more retarded than that ofscr-2, suggesting that it is the stronger allele (FIG. 2A). For bothalleles the aerial organs appeared similar to wild-type and the flowerswere fertile (FIGS. 2A and 2B). The progeny of backcrosses of scr-1 orscr-2 to wild-type plants segregated 3:1 for the root phenotype for bothalleles, indicating that each mutation is monogenic and recessive.

[0241] Analysis of transverse sections through the primary root ofseedlings revealed only a single cell layer between the epidermis andthe pericycle (FIG. 2C) instead of the normal radial organizationconsisting of cortex and endodermis (FIG. 2D). This radial organizationdefect was not limited to the primary root, but was also present insecondary roots (FIG. 2E) and in roots regenerated from calli (FIG. 2F).Occasionally defects were observed in the number of cells in theremaining cell layer (more than the invariant 8 found in wild-type).Abnormal placement or numbers of epidermal cells were also observed (seeFIG. 2E). These abnormalities were more frequently observed in scr-1than in scr-2. Nevertheless, organization of the mutant root closelyresembles that of wild-type except for the consistent reduction in thenumber of cell layers. Because the endodermis and cortex are normallygenerated by an asymmetric division of the cortex/endodermal initial,this indicates that the primary defect in scr is disruption of thisasymmetric division.

[0242] It has been shown that the radial organization defect in scr-1first appears in the developing embryo at the early torpedo stage andmanifests itself as a failure of the embryonic ground tissue to undergothe asymmetric division into cortex and endodermis (Scheres et al.,1995, Development 121:53-62). This defect extends the length of theembryonic axis which encompasses the embryonic root and hypocotyl. Otherembryonic tissues appear similar to wild-type (Scheres et al., 1995,Development 121:53-62). In seedling hypocotyls of the scarecrowphenotype, two cell layers instead of the normal three layers (twocortex and one endodermis) between epidermis and stele were found. Thiswould be the expected result of the lack of the division of theembryonic ground tissue. Similar results were obtained for scr-2. Hence,this mutant identifies a gene involved in the asymmetric division thatproduces cortex and endodermis from ground tissue in the embryonic rootand hypocotyl and from the cortex/endodermal initials in primary andsecondary roots.

[0243] 6.2.2. Characterization Of Cell Identity In SCR Roots

[0244] To understand the role of the Arabidopsis SCR gene in regulatingthis asymmetric division, it was necessary to determine the identity ofthe mutant cell layer. Tissue-specific markers were used to distinguishbetween several possibilities. The cell layer could have differentiatedattributes of either cortex or endodermis. Alternatively, it could havean undifferentiated, initial-cell identity or it could have a chimericidentity with differentiated attributes of both endodermis and cortex inthe same cell.

[0245] Transverse sections of scr-1 and scr-2 roots were assayed for thepresence of tissue-specific markers. The casparian strip, a depositionof suberin between radial cell walls, is specific to the endodermalcells and is believed to act as a barrier to the entry of solutes intothe vasculature (Esau, K. Anatomy of Seed Plants, New York: John Wiley &Sons, 1977, Ed. 2, pp. 1-550). Histochemical staining revealed thepresence of a casparian strip in the mutant cell layer (FIG. 3A, compareto wild-type, FIG. 3B). It is noted that in the vascular cylinder, thishistochemical stain also reveals the presence of lignin, indicating thepresence of differentiated xylem cells in mutant (FIG. 3A) and wild-type(FIG. 3B). Another marker of the differentiated endodermis is thearabinogalactan epitope recognized by the monoclonal antibody, JIM13(Knox et al., 1990, Planta 181:512-521). The mutant cell layer showedstaining with this antibody (FIG. 3C, compare with wild-type, FIG. 3B).As a positive control, the JIM7 antibody that recognizes pectin epitopesin all cell walls was used (FIGS. 3E and 7B). These results indicatethat the cell layer between the epidermis and the pericycle hasdifferentiated attributes of the endodermis.

[0246] As a marker for the cortex, the CCRC-M2 monoclonal antibody wasused. This antibody recognizes a cell wall oligosaccharide epitope,found only on differentiated cortex and epidermis cells. In sectionsfrom the differentiation zone of scr-1 and scr-2, both cortex andepidermal cells showed staining (FIG. 4A and 4B) that was similar tothat of wild-type (FIG. 4C). In scr-1, staining of both cell types wasapparent, but staining of cortex was somewhat weaker than wild-type. Thepositive control used the CCRC-M1 monoclonal antibody which recognizesan oligosaccharide epitope found on all cells (FIGS. 4D-F).

[0247] With the CCRC-M2 antibody an interesting difference was observedbetween the staining pattern of the mutants as compared to wild-type.The appearance of this epitope correlates with differentiation in thesetwo cell types. Normally, in sections close to the root tip there is nostaining. In sections higher up in the root, atrichoblasts (epidermalcells that do not make root hairs) stain. In sections from more matureroot tissue, all epidermal cells as well as cortex cells stain for thisepitope. In both scr-1 and scr-2, sections could be found in which allepidermal cells staines while there was little detectable staining ofcortex cells. Although not precisely identical the wild-type stainingpattern, the fact that the mutant cell layer clearly stains for thiscortex marker indicates that there are cortex differentiated attributesexpressed in these cells.

[0248] Taken together, these results indicate that the mutant cell layerhas differentiated attribues of both the endodermis and cortex. Thepossibility that there has been a simple deletion of a cell type, orthat the resulting cell type remains in an undifferentiated initial-likestage can be ruled out. This result is consistent with a role for thescr gene in regulating this asymmetric division rather than a role indirecting cell specification.

6.2.3. MOLECULAR CLONING OF THE SCR GENE

[0249] To further elucidate the function of the Arabidopsis SCR gene theinserted T-DNA sequences were used to clone the gene. Plant DNA flankingthe insertion site was obtained from scr-1 by plasmid rescue and used toisolate the corresdponding wild-type genomic DNA. Several cDNA cloneswere isolated from a library made from silique tissue. Comparision ofthe sequence of the longest CDNA and the corresponding genomic regionrevealed an open reading frame (ORF) interrupted by a single smallintron. (FIG. 5A). A potential TATA box and polyadenylation signal thatmatched the consensus sequences for plant genes were also identified(Joshi, C. P.., 1987, Nucl. Acids Res. 15:6643-6653) ; Heidecker &Messing, 1986, Ann. Rev. Plant Physiol. 37:439-466) ; Mogen et al.,1990, Plant Cell 2:1261-1272).

[0250] Comparison of the nucleotide sequence between the genomic cloneand the rescued plasmid placed the site of the T-DNA insertion in scr-1at codon 470 (FIGS. 5A and 5B). For scr-2, although no linkage was foundbetween the mutant phenotype and antibiotic resistance, DNA blot and PCRanalysis of antibiotic sensitive lines revealed the presence of T-DNAsequences that co-segregated with the mutant phenotype. The insertionposition in scr-2 was determined by cloning and sequencing the PCRproducts amplified from its genomic DNA using a combination of T-Dna andSCR specific primers at both sides of the insertion (FIG. 5B). In scr-2the T-DNA insertion point is at codon 605 (FIG. 5A and 5B). To verifylinkage between the cloned gene and the mutant phenotype, we identifiedthe chromosomal location of both the scr locus and the SCR gene. to mapthe scr locus, molecular markers were used on F2 progeny of crossesbetween scr-2 (ecotype Wassilewskija, Ws) and Colombia (Col) WT. Theseplaced the scr locus at the bottom of chromosome III, approximately 0.5cM away from each of the two closest markers available, cdc2b and BGL1(Konieczny and Ausubel, 1993, Plant j. 4:403-410). To map the SCR gene,we identified a polymorphism between Col and Landsberg (Ler) ecotypesusing the SCR probe b (FIG. 5B). Southern analysis of 25 recombinantinbred lines (Jarvis et al., 1994, Plant Mol. Biol. 24:685-687) mappedthe cloned gene to the same location as the SCR locus on chromosome III.

[0251] the determination of the molecular defects in two independentalleles and the co-localization of the cloned gene and the mutant locusconfirms that we have identified the SCR gene.

6.2.4. THE SCR GENE HAS MOTIFS THAT INDICATE IT IS A TRANSCRIPTIONFACTOR

[0252] The Arabidopsis SCR gene product is a 653 amino acid polypeptidethat contains several domains (FIG. 5B). The amino-terminus hashomopolymeric stretches of glutamine, serine, threonine, and prolineresideus, which account for 44% of the first 267 residues. Domains richin these residues have been shown to activate transcription and mayserve such a role in SCR (Johnson et al., 1993, J. Nutr. Biochem4:386-398). A charged region between residues 265 and 283 has similarityto the basic domain of the bZIP family of transcriptional regulatoryproteins (FIG. 5C) (Hurst, H. C., 1994, Protein Profile 1:123-168). Thebasick domains from several bZIP proteins have been shown to act asnuclear localization signals (Varagona et al., 1992, Plant Cell4:1213-1227), and this region in SCR may act similarly. This chargedregion is followed by a eucine heptad repeat (residues 291-322). Asecond leucine heptad repeat is found toward the carboxy-terminus(residues 436 to 473). As leucine heptad repeats have been demonstratedto mediate protein-protein interactions in other protens (Hurst, H. C.,1994, Protein Profile 1:123-168), the existence of these motifs suggeststhat SCR may function as a dimer or a multimer. The second leucineheptad repeat is followed by a small region rich in acidic residues,also present in a number of defined transcriptional activation domains(Johnson et al., 1993, J. Nutr Biochem 4:386-398). While each of thesedomains has been found within proteins that do not act astranscriptional regulators, the fact that all of them are found withinthe deduced SCR protein sequence indicates that SCR is a transcriptionalregulatory protein.

6.2.5. SCR IS A MEMBER OF A NOVEL PROTEIN FAMILY

[0253] The Arabidopsis SCR protein sequence was compared with thesequences in the available databases. Eleven expressed sequence tags(ESTs), nine from Arabidopsis, one from rice and one from maize, showedsignificant similarity to residues 394 to 435 of the SCR sequence, aregion immediately amino-terminal to the second leucine heptad repeat(FIGS. 15K-L). This region is designated the VHIID domain. Subsequentanalysis of these EST sequences has revealed that the sequencesimilarity extends throughout the entire known gene products. Thecombination and order of the motifs found in these sequences do not showsignificant similarity to the general structures of other establishedregulatory protein families (i.e., bZIP, zinc finger, MADS-domain, andhomeodomain), indicating that the SCR proteins comprise a novel family.

6.2.6. SCR IS EXPRESSED IN THE CORTEX/ENDODERMAL INITIALS AND IN THEENDODERMIS

[0254] RNA blot analysis revealed expression of SCR in Arabidopsissiliques, leaves and roots of wild-type plants (FIG. 6A). Nohybridization was detected to RNA from scr-1 plants (FIG. 6B, lane 2).This indicates that scr-1 has a reduced level of RNA expression and mayrepresent the null phenotye. Hybridization to RNA species larger thanthe normal size were detected in scr-2. This inidicates that abnormalSCR transcripts are made in this allele, suggesting that functional butpossibly altered proteins may be produced.

[0255] To determine if expression was localized to any particular celltype, RNA in situ was hybridization performed on sections of roottissue. In mature roots, expression was localized primarily to theendodermis (FIGS. 7A and 7B). Expression appeared to start very close toor within the cortex/endodermal initials and continue up the endodermalcell file as far as the section extended. Expression was also detectedin late-torpedo stage embryos in the endodermis throughout the embryonicaxis (FIG. 7C). Sense strand controls showed only backgroundhybridization (FIG. 7D).

[0256] To determine whether the localization of SCR RNA was regulated atthe transcriptional or post-transcriptional level, enhancer trap (ET)lines were prepared and examined in which the β-glucuronidase (uid-A orGUS) coding sequence with a minimal promoter was expressed in the rootendodermis. (See Section 7, infra). Restriction fragment lengthpolymorphisms were observed when DNA from one of these lines, ET199 andwild-type were probed with SCR. PCR and sequence analysis confirmed thatthe enhancer-trap construct had inserted approximately 1 kb upstream ofthe SCR start site and in the same orientation as that of SCRtranscription.

[0257] In mature roots, expression in ET199 whole mounts showed asimilar pattern to that of the in situ hybridizations, with thestrongest staining present in endodermal cells (FIG. 7E). Transversesections indicated that expression was primarily in endodermal cells inthe elongation zone (FIG. 7F). Longitudinal sections through themeristematic zone revealed that expression could be detected in thecortex/endodermal initial (FIG. 7G). Of particular interest was therestriction of expression to the endodermal daughter cell after thepericlinal division (FIG. 7G). This indicated that the expressionpattern observed in the in situ analysis was not due topost-transcriptional partitioning of SCR RNA. Rather, it suggests thatafter the periclinal division of the cortex/endodermis initial only oneof the two cells is able to transcribe SCR RNA.

[0258] 6.3. Discussion

[0259] 6.3.1. The SCR Gene Regulates An Asymmetric Division Required ForRoot Radial Organization

[0260] The formation of the cortex and endodermal layers in theArabidopsis root requires two asymmetric divisions. In the first, ananticlinal division of the cortex/endodermal initial generates two cellswith different developmental potentials. One will continue to functionas an initial, while the other undergoes a periclinal division togenerate the first cells in the endodermal and cortex cell files. Thissecond asymmetric division is eliminated in the scarecrow mutant,resulting in a single cell layer instead of two. The scr mutationappears to have little effect on any other cell divisions in the rootindicating that it is involved in regulating a single asymmetricdivision in this organ. Several other mutations have been characterizedthat appear to affect specific cell division pathways in Arabidopsis.These include knolle (kn) in which formation of the epidermis isimpaired (Lukowitz et al., 1996, Cell 84:61-71), wooden leg (wol) inwhich vascular cell division is defective (Scheres et al., 1995,Development 121:53-62) and fass (fs) in which there are supernumerarycortex and vascular cells (Scheres et al., 1995, Development 121:53-62);Torres Ruiz & Jurgens, 1994, Development 120:2967-2978). Only in thecase of scr and short-root (shr) mutants has it been shown that thedefect is in a specific asymmetric division.

[0261] Mutational analyses in several organisms have revealed that thegenes that regulate asymmetric divisions can be specific to a singletype of division or can affect divisions that are not clonally related(Horvitz & Herskowitz, 1992, Cell 68:237-255). In most cases, thesemutations result in the formation of two identical daughter cells withsimilar developmental potentials (Horvitz & Herskowitz, 1992, Cell68:237-255). Both resulting cells have the identity of one or the otherof the normal daughter cells, an example of which is the swi^(—)mutationin S. cerevisiae (Nasmyth et al., 1987, Cell 48:579-587). However, thereare also examples of mutations that result in the formation of chimericcell types such as the ham-1 mutation in C. elegans (Desai et al., 1988,Nature 336:638-646).

[0262] 6.3.2. SCR Involvement In Cell Specification Or Cell Division

[0263] Genes that regulate asymmetric cell divisions can be divided intothose that specify the differentiated fates of the daughter cells andthose that function to effect the division of the mother cell (Horvitz &Herskowitz, 1992, Cell, 68:237-255). The aberrant cell layer formed inthe scr mutant has differentiated features of both endodermal and cortexcells. Thus, scr is in the rare class of asymmetric division mutants inwhich a chimeric cell type is created. The ability to expressdifferentiated characteristics of cortex and endodermal cells impliesthat the differentiation pathways for both these cell types are intactand do not require the functional SCR gene. This indicates that SCR isinvolved primarily in regulating a specific cell division, and that thecorrect occurrence of this division can be unlinked from cellspecification. This is in contrast to the shr mutant, in which thepericlinal division of the cortex/endodermal initial also fails to occurand the resulting cell lacks endodermal markers (Benfey et al., 1993,Development 119:57-70) and has cortex attributes. A genetic analysis wasused to address the function of SHA and SCR in the asymmetric divisionof the cortex/endodermal initial. Placing mutants of each of these genesin a fs mutant background asked whether the supernumerary cell divisionscharacteristic of fs were sufficient to restore normal cell identities(Scheres et al., 1995, Development 121:53-62). In the shr,fs doublemutant there were additional cell layers but no endodermal, indicatingthat the SHR gene has a role in specifying cell identity. In the scr,fsdouble mutant no alteration in cell identity was observed as compared tofs (Scheres et al., 1995, Development 121:53-62). Taken together withthe cell marker analysis presented herein, these results are consistentwith a role for SCR in generating the division of the mother cell whilethe SHR gene may be involved in specifying the fate of the endodermaldaughter.

[0264] 6.3.3. A Role For SCR In Embryonic Development

[0265] At least one additional cell division appears to be affected inthe scr mutant. During embryonic development, the ground tissue does notdivide to form the endodermal and cortex layers of the embryonic rootand hypocotyl. As shown herein, expression of SCR was detected in theendodermal tissue throughout the embryonic axis shortly after thisdivision occurs. Thus, SCR may play a direct role in regulating boththis division and the division of the cortex/endodermal initial in theroot apical meristem. Alternatively, the radial organization establishedin the embryo may somehow act as a template that directs the division ofthe cortex/endodermal initial, thus perpetuating the pattern. This isconsistent with the finding in the scr mutant that the aberrant patternestablished in the embryo is perpetuated in the primary root. It is alsoconsistent with a recent study in which the daughter cells of thecortex/endodermal initial were laser ablated (van den Berg et al., 1995,Nature 378:62-65). When a single daughter cell was ablated, it wasreplaced by a cell that followed the normal asymmetric division pattern.When three adjacent daughter cells were ablated, the central initialdivided anticlinally but failed to perform the periclinal division (vanden Berg et al., 1995, Nature 378:62-65). This provided evidence thatinformation from mature cells is required for the correct divisionpattern of cortex/endodermal initials suggesting a “top down” transferof information. However, the absence of a cell layer in lateral rootsand callus-derived roots of the scr mutant suggests that embryo eventsare not unique in their ability to establish radial organization.Rather, these observations implicate SCR in regulating both embryonicand post-embryonic root radial organization.

[0266]6.3.4. Tissue-Specific Expression Of SCR Is Regulated At TheTranscriptional Level

[0267] Although not intending to be limited to any theory or explanationregarding the mechanism of SCR action, the cloning of the gene and theexpression pattern provide some clues as to the role of SCR in theregulation of a specific asymmetric division. The SCR gene is expressedin the cortex/endodermal initial, but immediately after division isrestricted to the endodermal lineage. A similar pattern is seen in theET199 enhancer trap line in which SCR regulatory elements are inproximity to a GUS gene, indicating that SCR restriction to theendodermal cell file is due to differential regulation of expression ofthe SCR gene in this cell and the first cell in the cortex file. Anothermarker line in which expression of GUS is detected only in the cortexdaughter cell provides a control for differential degradation of GUS RNAor protein. Thus, partitioning of SCR RNA as a means of achieving thissegregation of expression can be ruled out. What remains to bedetermined is whether this difference in transcriptional activity of thetwo daughter cells is due to internal polarity of the mother cell priorto division such that cytoplasmic determinants are unequallydistributed, or to external polarity that influences cell fate afterdivision. Since SCR is expressed prior to cell division, an attractivehypothesis is that it is involved in establishing polarity in thecortex/endodermal initial. The sequence of the SCR protein stronglysuggests that it acts as a transcription factor. Hence, it may act toregulate the expression of other genes essential for the establishmentof unequal division. Alternatively, it is conceivable that it could playa role in creating an external polarity that provides a signal to divideasymmetrically. Its expression in more mature endodermal cells isconsistent with a role in “top-down” signaling.

[0268] 6.3.5. A New Family of Transcriptional Regulators

[0269] Analysis of eighteen EST clones found in the GenBank databasereveals that the proteins they encode share a high degree of homologywith Arabidopsis SCR protein. See Table 1 and FIGS. 15A-S. Furthersequence analysis of the encoded proteins indicate that a high degree ofsequence similarity extends from at least the highly conserved VHIIDdomain to the carboxy-terminus of the gene products. Comparison of theamino termini of these proteins is precluded by the fact that the ESTsare incomplete. The high degree of similarity among these proteins, incombination with the motifs observed in the SCR protein (homopolymericmotifs, two leucine heptad repeats and a bZIP-like basic domain that mayalso function as a nuclear localization sequence) indicates that theseproteins form a novel class of regulatory proteins.

[0270] The insertion sites of the T-DNA in the two scr mutant allelesraised the possibility that the mutant phenotype was due to theproduction of truncated proteins. Northern blot analysis indicated SCRRNA is undetectable in scr-1. This suggests that the phenotype is eitherthe null, or due to highly reduced RNA expression. In scr-2, analteration in RNA size was detected which would be consistent with thepresence of a functional and possibly truncated protein. This couldprovide an explanation for the observation that scr-2 appears to be theweaker allele.

7. EXAMPLE 2

[0271] Enhancer Trap Analysis of Root Development

[0272] An enhancer trap system was used in order to provide a moredetailed molecular analysis of gene expression in lateral rootpatterning and development in Arabidopsis thaliana. A new collection ofmarker lines that express β-glucuronidase (GUS) activity in a cell-typespecific manner in each of the cells of the root was generated. Theselines allow differentiation of cells to be monitored based on molecularcharacteristics. One of these marker lines, ET199, resulted from theintegration of the GUS cassette in proximity to an SCR enhancer. Theresults described below demonstrate that transcriptional activation ofthe SCR gene plays an important role in root development in Arabidopsis,and that SCR gene transcriptional regulatory elements can express atransgene in a developmentally and tissue specific manner.

[0273] 7.1. Materials And Methods

[0274] 7.1.1. Plant Growth Conditions

[0275] Arabidopsis seeds from NO-O and Columbia ecotypes were sterilizedand sown on MS plates containing 4.5% sucrose. Plates were orientedvertically and maintained under 18 hours light, 6 hours dark cycle.

[0276] 7.1.2. Histology And Gus Staining

[0277] For observation of lateral roots, roots were removed from platesand infiltrated in 25% glycerol for several hours to overnight. Rootswere then mounted in 50% glycerol. Whole seedlings were stained for GUSactivity for up to three days in the following solution: 1×GUS buffer,20% methanol, 0.5 mg/ml X-Glu. Addition of methanol greatly improves thespecificity and reproducibility of staining. Staining solution was madefresh from a 10×buffer (1 M Tris pH7.5, 290 mg NaCl, 66 mg K₃Fe(CN)₆)that was stored for no more than one week. Stained roots were cleared-inglycerol and mounted as above. All samples were observed using Nomarskioptics on a Leitz Laborlux S microscope. Photographs were taken using aLeitz MPS52 camera, and images were scanned into Adobe Photoshop tocreate figures. In some cases the intensity of the blue color wasincreased.

[0278] 7.1.3. Construction Of Enhancer Trap Lines

[0279] Plant Cloning Vector (PCV) (Koncz et al., 1994, Specializedvectors for gene tagging and expression studies, in Plant MolecularBiology Manual, Gelvin & Schilperoort, eds., Vol. B2, pp. 1-2, KluoverAcademic Press, Dordrecht, The Netherlands) contains a Bam HI siteimmediately adjacent to the T-DNA right border sequence. Theβ-glucuronidase gene fused to the TATA region (−46 to 78) of the CaMV35S promoter was introduced into this site (Benfey et al., 1990, EMBO J.10 9:1677-1684). 350 transgenic lines were generated by Agrobacteriummediated root transformation (Marton & Browse, 1991, Plant Cell Reports10:235-239), and 4 independent lines from each transformant werescreened for GUS activity in the root.

[0280] 7.2. Results

[0281] 7.2.1. Differntiation In the LRP

[0282] The marker lines described above reflect patterns of geneexpression that are specific to individual root cell types. There are noreadily apparent mutant phenotypes in any of these lines. Therefore,they can be used to analyze the differentiation state of the cellsduring normal development of the lateral root primordial (LRP). If thereare stages at which the pericycle cells proliferate in the absence ofpatterning, it can be expected that all cells would be identical withnone expressing differentiated characteristics. In contrast,organization of the LRP would be reflected in differential patterns ofGUS gene expression, with certain cells beginning to turn ontranscription from differentiated cell-type specific promoters (i.e.,those that drive GUS expression in the enhancer trap lines).

[0283] The process of lateral root formation is divided into thefollowing seven stages:

[0284] Stage I: The LRP is first visible as a set of pericycle cellsthat are clearly shorter in length than their neighbors, havingundergone a series of anticlinal divisions. Laskowski et al., 1995, Dev.121:3303-3310 predict that there are approximately 4 founder pericyclecells involved. In the longitudinal plane, these divisions result in theformation of 8-10 small cells, which enlarge in a radial direction.

[0285] Stage II: A periclinal division occurs that divides the LRP intotwo layers (Upper Layer (UL) and Lower Layer (LL)). Not all the smallpericycle-derived cells appear to participate in this division --typically the most peripheral cells do not divide. Hence, as the UL andLL cells expand radially the domed shape of the LRP begins to appear.

[0286] Stage III: The UL divides periclinally, generating a three layerprimordium comprised of UL1, UL2 and LL. Again, some peripheral cells donot divide, creating peripheral regions that are one and two cell layersthick. This further emphasizes the domed shape of the LRP.

[0287] Stage IV: The LL divides periclinally, creating a total of fourcell layers (UL1, UL2, LL1, LL2). At this stage the LRP has penetratedthe parent endodermal layer.

[0288] Stage V: The central cells in LL2 undergo a number of divisionsthat push the overlying layers up and distort the cells in LL1. Thesedivisions are difficult to visualize at this stage, but clearly form aknot of mitotic activity. The LRP at this stage is midway through theparent cortex. The outer layer contains 10-12 cells.

[0289] Stage VI: This stage is characterized by several events. The fourcentral cells of UL1 divide periclinally. This division is particularlyuseful in identifying the median longitudinal plane in the enlargingLRP. At this point there are a total of twelve cells in UL1, four in themiddle that have undergone the periclinal division and four on eitherside. In addition, all but the most central cells of UL2 undergo apericlinal division. At this point the LRP has passed through the parentcortex layer and has penetrated the epidermis. The central cellsapparently derived from LL2 have a distinct elongated shapecharacteristic of vascular elements.

[0290] Stage VII: As the primordium enlarges it becomes difficult tocharacterize the divisions in the internal layers. However, the cells inthe outermost layer can still be seen very clearly. All of these cellsundergo a anticlinal division, resulting in 16 central cells (8 cells ineach of two layers) flanked by 8-10 cells on each side. We refer to thisas the 8-8-8 cell pattern. The LRP appears to be just about to emergefrom the parent root.

[0291] 7.2.2. Marker Lines

[0292] An enhancer trapping cassette was generated by fusing the GUScoding sequence to the minimal promoter of the 35S promoter from CaMV.This minimal promoter does not produce a detectable level of GUSexpression. However, its presence allows other upstream elements todirect GUS expression in a developmental and/or cell-specific manner(Benfey et al., 1990, EMBO J. 9:1677-1684). The use of a minimalpromoter instead of a promoterless construct allows GUS expression tooccur even if the enhancer trap cassette inserts at a distance from thecoding region. Since the insert does not have to be within thestructural gene, there are often no mutations generated in the enhancertrap lines. The minimal promoter:GUS construct was cloned immediatelyadjacent to the T-DNA right border sequence of PCV (Koncz et al., supra)and introduced into Arabidopsis. 350 independent lines were generatedand analyzed for GUS activity in the root. The following lines mostclearly define each cell type. All of the lines were generated throughenhancer trapping, as described herein, below, except for CorAX92(Dietrich et al., 1992, Plant Cell 4:1371-1382) and EpiGL2:GUS (Masucciet al., Dev. 122:1253-1260) which are transgenic plants that containcell-type specific promoters fused to the GUS gene.

[0293] Ste05-expresses GUS in the stele including the pericycle layerthroughout primary and lateral roots. At the root tip, staining becomesweaker in the elongation zone; therefore, it is likely that onlydifferentiated stele cells express GUS activity. Stelar GUS expressionis also seen in aerial parts of the plant.

[0294] End195-expresses GUS in the endodermis of primary and lateralroots. Staining can be seen most clearly in the cells in themeristematic region of the root, although overstaining shows that moremature cells also express some GUS activity. It appears that there is nostaining in the cortex/endodermal initial, but staining is evident inthe first daughter cell of this initial. GUS expression is also seen atthe base of young leaves and in the stipules.

[0295] ET199-expresses GUS in the endodermis of primary and lateralroots, again most clearly in cells in the meristematic region. UnlikeEnd195, staining in ET199 appears to continue down to thecortex/endodermal initial and, in younger roots, even into the cells ofthe quiescent center. Expression in the aerial parts of the plant isdetectable in the young leaf primordia.

[0296] CorAX92-This line was generated by fusing the 5′ and 3′ sequencesfrom a cortex specific gene isolated from oilseed rape to the GUSreporter gene (Dietrich et al., Plant Cell 4:1371-1382). Expression islimited to the cortex layer, extending to but not including thecortex/endodermal initial. Staining is also apparent in the petioles andleaf blades of expanded leaves.

[0297] EpiGL2:GUS-This line was generated by fusing the GL2 promoter tothe GUS gene (Masucci et al., Dev. 122:1253-1260). Expression is seen inthe non-hair forming epidermal cells (atrichoblasts). Staining is seennear the root tip, but it is difficult to determine if it includes theepidermal initial. Staining is also seen in the trichomes, leafprimordia, and the epidermis of the hypocotyl and leaf petioles.

[0298] CRC219-This line shows staining in the columella root cap only.

[0299] LRC244-This line shows staining in the lateral root cap only.

[0300] RC162-This line shows staining in both the lateral and columellaroot caps.

[0301] Two marker lines show differential staining at very early stagesof LRP development. One of these, ET199, presents a complex and dynamicpattern of expression. Staining is first apparent at stage II in onlythe four central cells of the UL. At stage III staining is strongest inthe central cells of UL2. As the LRP reaches stage V the stainingremains strongest in the central 2-4 cells of UL2. By stage VI stainingalso begins to extend into the newly formed endodermal layer, andstaining in both the central cells and endodermis persists beyondemergence of the lateral root.

[0302] Another line, LRB10 (lateral root base), does not express GUS inthe primary root tip. Staining in the LRP is seen at stage I, and atstage II all the cells of the UL and LL are stained. However, by stageIV and V only the cells at the periphery of the LRP are still expressingGUS. As the LRP develops, these cells continue to stain, although lessintensely, resulting in a ring of GUS expressing cells at the base ofthe LR.

[0303] LRB10 and ET199 clearly demonstrate non-identity between thecells at very early stages, stage IV in the case of LRB10 and within theUL at stage II in ET199. In addition, although it is difficult toidentify the nature of the cells that correspond to the observedstaining pattern in LRB10 and the early staining cells of ET199,post-emergent lateral roots show analogous staining in these lines,suggesting that the stained cells are already expressing markers thatreflect their differentiated cell fates. Hence, these observationssuggest a very early onset of differentiation in the cells of the LRP.

[0304] 7.2.3. ET199 Provides Evidence For The Role Of SCR In PlantDevelopment

[0305] Fortuitously, it was discovered that the GUS cassette in ET199described Section 7.2.2, above, is situated approximately 1 kb upstreamfrom the SCR gene. The SCR cDNA was labelled and used to probe genomicDNA from WT and ET199 plants. The band pattern seen in the Southern wascompletely consistent with a T-DNA inserted 1 kb upstream of theputative SCARECROW start site. Subsequently, a DNA fragment was PCRamplified using a primer within the T-DNA and a primer within SCARECROW.The size of this fragment was also consistent with the predictedinsertion site. Partial sequencing of the PCR fragment confirmed thepresence of SCARECROW sequence. Mutants in the SCR gene are completelylacking one of the radial layers between the epidermis and pericycle inboth primary and lateral roots, due to the absence of specific celldivision during embryogenesis and of the cortex/endodermal initialduring post-embryonic growth. The expression pattern (described inSection 7.2.2., above) that was observed in the central cells of thedeveloping LRP of ET199 provide strong evidence that the cells in thisregion are involved in the establishment of the meristematic initials.More importantly, these results demonstrate that transcriptionalactivation of the SCR gene plays a major role in the development of theArabidopsis LRP. Furthermore, these results demonstrate that a transgenecan be expressed under the control of SCR gene transcriptionalregulatory elements in a developmental and tissue-specific manner.

8. EXAMPLE 3

[0306] Activity of Arabidopsis Scr Promoter in Transgenic Roots

[0307] The expression pattern of Arabidopsis SCR has been determined byanalysis of an enhancer trap line, ET199, in which a GUS coding regionwith a minimal promoter was fortuitously inserted 1 kb upstream of theSCR coding region (see supra). In ET199 plants, GUS expression isdetected in the endodermis, endodermal initials and sometimes in thequiescent center (QC) of the root. See supra and Malamy and Benfey,1997, Dev. 124:33-44. This expression pattern of SCR in the primary roothas been confirmed by in situ analysis (See supra and Di Laurenzio etal., 1996, Cell 86:423-433).

[0308] The following experiments demonstrate that 2.5 kb of 5′ sequenceupstream of the Arabidopsis SCR coding region is sufficient to conferSCR expression pattern to a heterologous gene. The 5′ sequence used inthese studies starts from the Hind III site approximately 2.5 kbupstream of the ATG initiation site and extends 3′ downstream to thebase pair immediately upstream of the ATG initiation site (see FIG. 14).This 5′ sequence was fused to a GUS coding sequence. The resulting SCRpromoter::GUS construct was incorporate into an Agrobacterium vector,which was used to transform and generate transgenic roots using standardprocedures.

[0309] A large number of roots were regenerated. They show GUS stainingpattern that is similar to the SCR expression pattern in ET199 plants(FIG. 19, Panel f). Since organs regenerated from callus often have anabnormal morphology, transgenic roots were transferred to liquidculture. Roots grown in liquid culture appeared morphologically normaland showed GUS expression in the endodermis, endodermal initial and QC(FIG. 19, Panel g), similar to the expression pattern of SCR seen in theenhancer trap line ET199. These results indicate that the 2.5 kb regionupstream of the SCR start site is sufficient to confer the SCRexpression pattern in the root.

[0310] The expression of the SCR promoter::GUS construct was alsoexamined in scr mutant background. The scr mutant has an altered rootorganization (see, supra). Whereas the wild-type root of Arabidopsis hasfour distinct cell layers surrounding the vascular tissue, the roots ofscr mutant have only three.

[0311] Transgenic roots of the scr mutant were generated that containeda SCR promoter::GUS construct. As in the wild-type, a large number oftransgenic roots were formed that had detectable GUS expression (FIG.20, Panel a). These roots were shorter than wild-type regenerated roots,consistent with the shorter root phenotype of the scr mutant.

[0312] Additional transgenic root experiments demonstrated that the SCRgene under control of its own promoter can rescue the scr mutantphenotype. Transgenic scr roots were generated that contained the fulllength SCR gene under the control of its own promoter. The length oftransgenic roots containing the construct were longer than those of thescr mutant, indicating that the introduced SCR gene partially rescuedthe mutant. Whereas scr regenerated roots that carried the SCRpromoter::GUS construct were very short (FIG. 21, Panel a; and FIG. 20,Panel a), roots transformed with the SCR promoter and coding region werenoticeably longer (FIG. 21, Panel b). The difference was even moreobvious in liquid culture, in which scr mutant roots remained short(FIG. 21, Panel c), while SCR gene complemented scr mutant roots werelong and resembled wild-type roots (FIG. 21, Panel d).

[0313] Anatomical studies of the regenerated roots confirmed the abilityof the SCR promoter: :SCR gene construct to rescue the scr mutantphenotype. Whereas regenerated roots of scr mutant were missing aninternal layer (FIG. 21, Panel e), the scr mutant roots that weretransformed with the SCR promoter: :SCR gene construct had a radialorganization that resembled wild-type root (FIG. 21, Panel f).

9. EXAMPLE 4

[0314] Isolation Scr Sequences Using Pcr-Cloning Strategy

[0315] Based on the comparison of the sequences of SCR paralogs inArabidopsis, degenerate primers SCR3AII, SCR5AII and SCR5B were designedand used in PCR amplification of SCR sequences from genomic DNA ofvarious plant species. The amplification was performed according tocondition described in Section 5.1.1., supra, using DNA isolated frommaize plants grown from a commercial seed mixture. Amplificationproducts (104 bp fragment for the SCR5B+SCR3AII primer combination; 146bp fragment for the SCR5AII+SCR3AII primer combination) were obtained,and each cloned into a T/A vector (Invitrogen, San Diego, Calif.) andsequenced. Two of the three different types of clones obtained haddeduced amino acid sequences that were very similar to a part of theArabidopsis SCR protein (i.e., approximately 90% identity), suggestingthat they represent parts from two different alleles of the maize SCRgene (i.e., ZCR gene). The two clones each had only two conservativechanges in their nucleotide sequence.

[0316] The 146 bp amplification product, ZmScl1, was subsequently usedas a probe for screening of a genomic library generated in lambdaBlueSTAR vector (NOVAGEN) from maize (HiII line) genomic DNA. Thescreening was performed according to the standard procedures describedin Genius™ System User's Guide For MembraneHybridization(Boehringer-Mannheim): The probe was a single-strand DNAmolecule corresponding to the ZmScl1 fragment produced by PCR (Genius,Boehringer-Mannheim). Hybridization was performed according torecommendations of the manufacturer′s manual (Boehringer-Mannheim).Prehybridization was for 2 hr in 50% formamide hybridization solution at42° C. Hybridization was overnight at 42° C. with 200 ng/ml probeconcentration. Filters were washed twice at room temperature in 2×SSC,0.1% SDS for 5 min, and for stringent washing at 65° C. in 0.5×SSC,0.1%SDS twice for 15 min.

[0317] A positive clone was identified. The clone contained a 13 kbinsert, which was subcloned into a plasmid vector. The resulting plasmidwas designated pZCR. A 5 kb Eco RI fragment containing the maize SCR(ZCR) sequence was subcloned and sequenced. The nucleotide sequence ofthe region containing a partial ZCR coding sequence is shown in FIG. 17Aand the corresponding deduced amino acid sequence is shown in FIG. 17B.The ZCR protein contain a segment that is highly homologous to acorresponding segment in the Arabidopsis SCR protein (FIG. 17B). Thissegment is flanked by segments of low homology. Thus, it is possiblethat the genomic clone of ZCR is a composite clone, containing sequencesthat are not ZCR sequences.

[0318] The deduced ZCR protein sequence was aligned with that ofArabidopsis SCR protein. The comparison revealed new conserved sites inthe SCR coding sequence which were used to design new, more specific PCRprimers (i.e., 1F, 1R, and 4R) for use in amplification of SCR sequencesfrom yet other plant species.

[0319] Using combinations of primers 1F+1 R and 1F+4R, PCR amplificationwere performed as described in section 5.1.1.. Two DNA of expected sizewere obtain from soybean: a 247 bp DNA from the 1F+1 R primercombination and a 379 bp DNA from the 1F+4R primer combination. A DNA ofexpected size (247 kb) was obtained from carrot and spruce when theirgenomic DNA was amplified using 1F+4R primer combination. The nucleotidesequences of the 379 kb soybean DNA (SRPg1 ), the 247 kb DNA from carrot(SRPd1) and spruce (SRPp1) are shown in FIGS. 16K-M. The correspondingdeduced amino acid sequences of these amplified sequences are shown inFIG. 18. Comparison of these partial SCR coding sequences indicate thisapproach isolated DNA sequences that encode SCR proteins with amino acidsequences that are very similar but not identical to a segment ofArabidopsis SCR protein (see FIG. 18).

10. EXAMPLE 5

[0320] Expression Pattern of Maize ZCR Gene In Root Tissue

[0321] These experiments examined the expression pattern of ZCR in theprimary root and quiescent centers of maize root. The expression patternwas determined by in situ hybridization using a ZCR RNA probe,corresponding to an amino acid segment region that is highly homologousto a corresponding segment of the Arabidopsis SCR protein. Theexperiment was carried out as follows. Restriction fragments containingthe maize ZCR sequence were isolated from pZCR and subcloned into apBluescript vector for in vitro transcription. The probe was synthesizedusing conditions described in the Genius Dig RNA labeling kit. ThepBluescript plasmid was linearized, and 1 μg was used as a template tosynthesize digoxigenin-labeled RNA using the T7 polymerase. The RNAprobe was subjected to mild alkali hydrolysis by heated at 600° C. for 1hr in 100 mM carbonate buffer (pH 10.2) to yield a probe size ofapproximately 0.15 kb. Probe concentration for hybridization wasoptimized at 1 μg/ml/kb. In situ hybridization of root tips from 48 to72 hr-old maize seedlings or excised quiescent centers (QCs) of rootswere carried out following procedures described in Section 6.1.6.,supra.

[0322] The results show that ZCR expression in maize primary roots islocalized to a file of cells that is identified as the endodermal layer.The expression pattern continues in a single uninterrupted file throughthe QC which consists of approximately 1000-1500 cells (FIG. 22).

[0323] In two-week old regenerating QCs, ZCR expression is found in afile of cells extending through the newly formed apex. Thus, theregenerated roots exhibits a ZCR expression pattern that is similar tothat seen in the primary root, even though the root apex does notcontain the normal arrangement of cell files at this stage.

[0324] ZCR expression during regeneration of the root apex was alsoexamined. In the initial stages of regeneration, cell proliferationoccurs to fill in the removed tissue and begins to regenerate the basicshape of the root tip. All cells on the blunt edge of the root appearsto contribute to the new population of cells. The ZCR expression patternindicates that molecular signals are differentially present in thesecells at an early stage in regeneration. The gene appears to bediagnostic of cells that are preparing to undergo asymmetrical divisionin order to re-establish the normal organization of the root apex fromthe large undifferentiated cells. The results indicate that ZCRexpression is required for pattern formation since it is expressed priorto the generation of any specific anatomical pattern in the newly formednot tissue.

11. EXAMPLE 6

[0325] Expression Pattern of ZCR Gene in Soybean Roots and Root Nodules

[0326] SCR expression in soybean roots and nodules was examined using insitu hybridization with a SCR probe. The procedure used were asdescribed in Sections 6.1.6. and 11.

[0327] In primary roots, SCR is expressed in the endodermis. Expressionwas also found in cells at the root tip that are located at the distalend of the endodermal cell files. In soybean nodules, expression of SCRwas detected in the peripheral tissue at the site of developing vascularstrands. At later stages of vascular development within the nodule, SCRexpression was found flanking the vascular tissue. These resultsindicate that SCR is involved in regulating vascularization in thenodule by contributing to the radial organization that is required togenerate endodermis. These findings indicate that SCR promoter may beused to express proteins in a highly tissue-specific manner in soybeannodules. One application is to use SCR promoter to engineer nodulesthrough production of components in a tissue-specific manner. Anotherapplication is that modification of the expression of SCR could enhancenodule activity by improving vascularization and/or the number ofendodermal layers.

12. EXAMPLE 7

[0328] Scr Expression Affects Gravitropism of Aerial Structure

[0329] In addition to being defective in specific embryonic andpostembryonic meristematic divisions, both the scr and the shr mutantshave shoots that exhibit severely defective gravitropism.Complementation analysis showed that scr is allelic to a sgr (shootgravitropism) mutant, sgr1 . Four mutant alleles of SCR (i.e., scr1,scr2, sgr1-1 and sgr1-2) have been identified. All four of these mutantshave normal root gravitropism and defective shoot gravitropism.

[0330] Etiolated hypocotyls of scr mutants placed on their sides do notrespond to gravity even after 3 hr. Similar behaviors were observed withthe inflorescence stems of sgr1-1 mutant, which do not curve upwardseven after two days on their sides. In contrast, the roots of theseplants respond rapidly to the change in orientation with the samekinetics as the wild type. Thus, mutations in the SCR gene lead to aradial pattern deficiency in the root but have no effect on rootgravitropism.

[0331] Comparable results were also obtained for shr roots and forhypocotyls and inflorescence stems, i.e., data indicate that shr showsnormal root gravitropism but almost no stem gravitropism.

[0332] 13. Deposit Of Microorganisms

[0333] The following microorganisms have been deposited in accordancewith the terms of the Budapest Treaty with the American Type CultureCollection; 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., on thedates indicated: Micro- Accession organism Clone No. Date DH5α pGEX-2TK⁺98031 April 26, 1996 (pLIG 1-3/Sac + MOB1Sac) DH5α pNYH1 (Zm-sc11b)98032 April 26, 1996 DH5α pNYH2 (Zm-sc11) 98033 April 26, 1996 DH5αpNYH3 (Zm-sc12) 98034 April 26, 1996 DH5α pZCR April 18, 1997

[0334] Although the invention is described in detail with reference tospecific embodiments thereof, it will be understood that variationswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings suchmodifications are intended to fall within the scope of the appendedclaims.

[0335] Various publications are cited herein, each of the disclosures ofwhich is incorporated by reference in its entirety.

1 79 1 2163 DNA Arabidopsis thaliana 1 ccttatttat aaccatgcaa tctcacgaccaacaaccctt caatctccat ggcggaatcc 60 ggcgatttca acggtggtca acctcctcctcatagtcctc tgagaacaac ttcttccggt 120 agtagcagca gcaacaaccg tggtcctcctcctcctcctc ctcctccttt agtgatggtg 180 agaaaaagat tagcttccga gatgtcttctaaccctgact acaacaactc ctctcgtcct 240 cctcgccgtg tctctcacct tcttgactccaactacaata ctgtcacacc acaacaacca 300 ccgtctctta cggcggcggc tactgtatcttctcaaccaa acccaccact ctctgtttgt 360 ggcttctctg gtcttcccgt ttttccttcagaccgtggtg gtcggaatgt tatgatgtcc 420 gtacaaccaa tggatcaaga ctcttcatcttcttctgctt cacctactgt atgggttgac 480 gccattatca gagaccttat ccattcctcaacttcagtct ctattcctca acttatccaa 540 aacgttagag acattatctt cccttgtaacccaaatctcg gtgctcttct tgaatacagg 600 ctccgatctc tcatgctcct tgatccttcctcttcctctg acccttctcc tcaaactttc 660 gaacctctct atcagatctc caacaatccttctcctccac aacagcaaca gcagcaccaa 720 caacaacaac aacagcataa gcctcctcctcctccgattc agcagcaaga aagagaaaat 780 tcttctaccg atgcaccacc gcaaccagagacagtgacgg ccactgttcc cgccgtccaa 840 acaaatacgg cggaggcttt aagagagaggaaggaagaga ttaagaggca gaagcaagac 900 gaagaaggat tacaccttct cacattgctgctacagtgtg ctgaagctgt ctctgctgat 960 aatctcgaag aagcaaacaa gcttcttcttgagatctctc agttatcaac tccttacggg 1020 acctcagcgc agagagtagc tgcttacttctcggaagcta tgtcagcgag attactcaac 1080 tcgtgtctcg gaatttacgc ggctttgccttcacggtgga tgcctcaaac gcatagcttg 1140 aaaatggtct ctgcgtttca ggtctttaatgggataagcc ctttagtgaa attctcacac 1200 tttacagcga atcaggcgat tcaagaagcatttgagaaag aagacagtgt acacatcatt 1260 gacttggaca tcatgcaggg acttcaatggcctggtttat tccacattct tgcttctaga 1320 cctggaggac ctccacacgt gcgactcacgggacttggta cttccatgga agctcttcag 1380 gctacaggga aacgtctttc ggatttcacagataagcttg gcctgccttt tgagttctgc 1440 cctttagctg agaaagttgg aaacttggacactgagagac tcaatgtgag gaaaagggaa 1500 gctgtggctg ttcactggct tcaacattctctttatgatg tcactggctc tgatgcacac 1560 actctctggt tactccaaag gtaaaataaacattaccttt taatcactct ttatctataa 1620 attattttaa gattatatag gaaagatatgttctaaaaag ctggcttttt tggttaatga 1680 ttggggaatg aacagattag ctcctaaagttgtgacagta gtggagcaag atttgagcca 1740 cgctggttct ttcttaggaa gatttgtagaggcaatacat tactactctg cactctttga 1800 ctcactggga gcaagctacg gcgaagagagtgaagagaga catgtcgtgg aacagcagct 1860 attatcgaaa gagatacgga atgtattagcggttggagga ccatcgagaa gcggtgaagt 1920 gaagtttgag agctggaggg agaaaatgcaacaatgtggg tttaaaggta tatctttagc 1980 tggaaatgca gctacacaag cgactctactgttgggaatg tttccttcgg atggttacac 2040 tttggttgat gataatggta cacttaagcttggatggaaa gatctttcgt tactcactgc 2100 ttcagcttgg acgcctcgtt cttagttttcttctcctttt tcacaaacaa tgtgcccata 2160 aat 2163 2 653 PRT Arabidopsisthaliana 2 Met Ala Glu Ser Gly Asp Phe Asn Gly Gly Gln Pro Pro Pro HisSer 1 5 10 15 Pro Leu Arg Thr Thr Ser Ser Gly Ser Ser Ser Ser Asn AsnArg Gly 20 25 30 Pro Pro Pro Pro Pro Pro Pro Pro Leu Val Met Val Arg LysArg Leu 35 40 45 Ala Ser Glu Met Ser Ser Asn Pro Asp Tyr Asn Asn Ser SerArg Pro 50 55 60 Pro Arg Arg Val Ser His Leu Leu Asp Ser Asn Tyr Asn ThrVal Thr 65 70 75 80 Pro Gln Gln Pro Pro Ser Leu Thr Ala Ala Ala Thr ValSer Ser Gln 85 90 95 Pro Asn Pro Pro Leu Ser Val Cys Gly Phe Ser Gly LeuPro Val Phe 100 105 110 Pro Ser Asp Arg Gly Gly Arg Asn Val Met Met SerVal Gln Pro Met 115 120 125 Asp Gln Asp Ser Ser Ser Ser Ser Ala Ser ProThr Val Trp Val Asp 130 135 140 Ala Ile Ile Arg Asp Leu Ile His Ser SerThr Ser Val Ser Ile Pro 145 150 155 160 Gln Leu Ile Gln Asn Val Arg AspIle Ile Phe Pro Cys Asn Pro Asn 165 170 175 Leu Gly Ala Leu Leu Glu TyrArg Leu Arg Ser Leu Met Leu Leu Asp 180 185 190 Pro Ser Ser Ser Ser AspPro Ser Pro Gln Thr Phe Glu Pro Leu Tyr 195 200 205 Gln Ile Ser Asn AsnPro Ser Pro Pro Gln Gln Gln Gln Gln His Gln 210 215 220 Gln Gln Gln GlnGln His Lys Pro Pro Pro Pro Pro Ile Gln Gln Gln 225 230 235 240 Glu ArgGlu Asn Ser Ser Thr Asp Ala Pro Pro Gln Pro Glu Thr Val 245 250 255 ThrAla Thr Val Pro Ala Val Gln Thr Asn Thr Ala Glu Ala Leu Arg 260 265 270Glu Arg Lys Glu Glu Ile Lys Arg Gln Lys Gln Asp Glu Glu Gly Leu 275 280285 His Leu Leu Thr Leu Leu Leu Gln Cys Ala Glu Ala Val Ser Ala Asp 290295 300 Asn Leu Glu Glu Ala Asn Lys Leu Leu Leu Glu Ile Ser Gln Leu Ser305 310 315 320 Thr Pro Tyr Gly Thr Ser Ala Gln Arg Val Ala Ala Tyr PheSer Glu 325 330 335 Ala Met Ser Ala Arg Leu Leu Asn Ser Cys Leu Gly IleTyr Ala Ala 340 345 350 Leu Pro Ser Arg Trp Met Pro Gln Thr His Ser LeuLys Met Val Ser 355 360 365 Ala Phe Gln Val Phe Asn Gly Ile Ser Pro LeuVal Lys Phe Ser His 370 375 380 Phe Thr Ala Asn Gln Ala Ile Gln Glu AlaPhe Glu Lys Glu Asp Ser 385 390 395 400 Val His Ile Ile Asp Leu Asp IleMet Gln Gly Leu Gln Trp Pro Gly 405 410 415 Leu Phe His Ile Leu Ala SerArg Pro Gly Gly Pro Pro His Val Arg 420 425 430 Leu Thr Gly Leu Gly ThrSer Met Glu Ala Leu Gln Ala Thr Gly Lys 435 440 445 Arg Leu Ser Asp PheThr Asp Lys Leu Gly Leu Pro Phe Glu Phe Cys 450 455 460 Pro Leu Ala GluLys Val Gly Asn Leu Asp Thr Glu Arg Leu Asn Val 465 470 475 480 Arg LysArg Glu Ala Val Ala Val His Trp Leu Gln His Ser Leu Tyr 485 490 495 AspVal Thr Gly Ser Asp Ala His Thr Leu Trp Leu Leu Gln Arg Leu 500 505 510Ala Pro Lys Val Val Thr Val Val Glu Gln Asp Leu Ser His Ala Gly 515 520525 Ser Phe Leu Gly Arg Phe Val Glu Ala Ile His Tyr Tyr Ser Ala Leu 530535 540 Phe Asp Ser Leu Gly Ala Ser Tyr Gly Glu Glu Ser Glu Glu Arg His545 550 555 560 Val Val Glu Gln Gln Leu Leu Ser Lys Glu Ile Arg Asn ValLeu Ala 565 570 575 Val Gly Gly Pro Ser Arg Ser Gly Glu Val Lys Phe GluSer Trp Arg 580 585 590 Glu Lys Met Gln Gln Cys Gly Phe Lys Gly Ile SerLeu Ala Gly Asn 595 600 605 Ala Ala Thr Gln Ala Thr Leu Leu Leu Gly MetPhe Pro Ser Asp Gly 610 615 620 Tyr Thr Leu Val Asp Asp Asn Gly Thr LeuLys Leu Gly Trp Lys Asp 625 630 635 640 Leu Ser Leu Leu Thr Ala Ser AlaTrp Thr Pro Arg Ser 645 650 3 23 PRT Arabidopsis thaliana 3 Pro Ala ValGln Thr Asn Thr Ala Glu Ala Leu Arg Glu Arg Lys Glu 1 5 10 15 Glu IleLys Arg Gln Lys Gln 20 4 23 PRT Saccharomyces sp. 4 Leu Lys Arg Ala ArgAsn Thr Glu Ala Ala Arg Arg Ser Arg Ala Arg 1 5 10 15 Lys Leu Gln ArgMet Lys Gln 20 5 23 PRT Arabidopsis thaliana 5 Arg Arg Leu Ala Gln AsnArg Glu Ala Ala Arg Lys Ser Arg Leu Arg 1 5 10 15 Lys Lys Ala Tyr ValGln Gln 20 6 23 PRT Mus musculus 6 Ile Arg Arg Glu Arg Asn Lys Met AlaAla Ala Lys Cys Arg Asn Arg 1 5 10 15 Arg Arg Glu Leu Thr Asp Thr 20 723 PRT Homo sapiens 7 Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser LysCys Arg Lys Arg 1 5 10 15 Lys Leu Glu Arg Ile Ala Arg 20 8 23 PRT Homosapiens 8 Val Arg Leu Met Lys Asn Arg Glu Ala Ala Arg Glu Cys Arg ArgLys 1 5 10 15 Lys Lys Glu Tyr Val Lys Cys 20 9 23 PRT Zea mays 9 Lys ArgLys Glu Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Tyr Arg 1 5 10 15 LysAla Ala His Leu Lys Glu 20 10 23 PRT Zea mays 10 Met Arg Gln Ile Arg AsnArg Asp Ser Ala Met Lys Ser Arg Glu Arg 1 5 10 15 Lys Lys Ser Tyr IleLys Asp 20 11 23 PRT Oryza sp. 11 Arg Arg Met Val Ser Asn Arg Glu SerAla Arg Arg Ser Arg Lys Lys 1 5 10 15 Lys Gln Ala His Leu Ala Asp 20 1243 PRT Arabidopsis thaliana 12 Ala Phe Glu Lys Glu Asp Ser Val His IleIle Asp Leu Asp Ile Met 1 5 10 15 Gln Gly Leu Gln Trp Pro Gly Leu PheHis Ile Leu Ala Ser Arg Pro 20 25 30 Gly Gly Pro Pro His Val Arg Leu ThrGly Leu 35 40 13 43 PRT Arabidopsis thaliana 13 Ala Val Lys Asn Glu SerPhe Val His Ile Ile Asp Phe Gln Ile Ser 1 5 10 15 Gln Gly Gly Gln TrpVal Ser Leu Ile Arg Ala Leu Gly Ala Arg Pro 20 25 30 Gly Gly Pro Pro AsnVal Arg Ile Thr Gly Ile 35 40 14 43 PRT Arabidopsis thaliana 14 Ala MetGlu Gly Glu Lys Met Val His Val Ile Asp Leu Asp Ala Ser 1 5 10 15 GluPro Ala Gln Trp Leu Ala Leu Leu Gln Ala Phe Asn Ser Arg Pro 20 25 30 GluGly Pro Pro His Leu Arg Ile Thr Gly Val 35 40 15 29 PRT Arabidopsisthaliana 15 Ala Ile Lys Gly Glu Glu Glu Val His Ile Ile Asp Phe Asp IleAsn 1 5 10 15 Gln Gly Asn Gln Tyr Met Thr Leu Ile Arg Ser Ile Ala 20 2516 26 PRT Oryza sp. VARIANT (1)...(26) Xaa = Any Amino Acid 16 Ile HisVal Ile Asp Phe Xaa Leu Gly Val Gly Gly Gln Trp Ala Ser 1 5 10 15 PheLeu Gln Glu Leu Ala His Arg Arg Gly 20 25 17 36 PRT Zea mays VARIANT(1)...(36) Xaa = Any Amino Acid 17 Val His Ile Ile Xaa Phe Xaa Leu MetGln Gly Leu Gln Trp Pro Ala 1 5 10 15 Leu Met Asp Val Phe Ser Ala ArgLys Gly Gly Pro Pro Lys Leu Arg 20 25 30 Ile Thr Gly Ile 35 18 1085 DNAArabidopsis thaliana misc_feature (1)...(1085) n = A,T,C or G 18ggcacgagcc caacgggtcc tgagcttctt acttatatgc atatcttgta tgaagcctgc 60ccttatttca aattcggtta tgaatctgct aatggagcta tagctgaagc tgtgaagaac 120gaaagttttg tgcacattat cgatttccag atttctcaag gtggtcaatg ggtgagtttg 180atccgtgctc ttggtgctag acctggtgga cctccgaacg ttaggataac gggaattgat 240gatccgagat catcgtttgc tcgtcaagga ggacttgagt tagttggaca aagacttggg 300aagctagctg aaatgtgcgg tgttccgttt gagttccatg gagctgcttt atgctgcacg 360gaagtcgaaa tcgagaagct aggagttaga aatggagaag cgctcgcggt taacttcccg 420cttgttcttc accacatgcc tgatgagagt gtaactgtgg agaatcacag agatagattg 480ttgagattgg tcaaacactt gtcaccaaac gttgtgactc tggttgagca agaagcgaat 540acaaacactg cgccgtttct tccccggttt gtcgagacaa tgaaccatta cttggcagtt 600ttcgaatcaa tagatgtgaa actcgctaga gatcacaagg aaaggatcaa tgttgagcag 660cattgtttgg ctagagaggt tgtgaatctt atagcttgtg aaggtgttga aagagaagag 720aggcacgagc cactagggaa atggaggtct cggtttcaca tggcgggatt taaaccgtat 780cctttgagct cgtatgtgaa cgcaacaatc aaaggattgc ttgagagtta ttcagagaag 840tatacacttg aagaaagaga tggagcattg tatttaggat ggaagaatca acctcttatc 900acttcttgtg cttggaggta actaataaaa accttgttcg gtttcagaag agattagaaa 960cttcttttaa agtttgcaga atctgtttgt aaaagtaaaa ctcatgcatg atccgnagga 1020acaagttgtc aaatgttgta gtagtaagtg atatgttgat gacccaaaaa aaaaaaaaaa 1080aaaaa 1085 19 306 PRT Arabidopsis thaliana 19 Gly Thr Ser Pro Thr GlyPro Glu Leu Leu Thr Tyr Met His Ile Leu 1 5 10 15 Tyr Glu Ala Cys ProTyr Phe Lys Phe Gly Tyr Glu Ser Ala Asn Gly 20 25 30 Ala Ile Ala Glu AlaVal Lys Asn Glu Ser Phe Val His Ile Ile Asp 35 40 45 Phe Gln Ile Ser GlnGly Gly Gln Trp Val Ser Leu Ile Arg Ala Leu 50 55 60 Gly Ala Arg Pro GlyGly Pro Pro Asn Val Arg Ile Thr Gly Ile Asp 65 70 75 80 Asp Pro Arg SerSer Phe Ala Arg Gln Gly Gly Leu Glu Leu Val Gly 85 90 95 Gln Arg Leu GlyLys Leu Ala Glu Met Cys Gly Val Pro Phe Glu Phe 100 105 110 His Gly AlaAla Leu Phe Cys Thr Glu Val Glu Ile Glu Lys Leu Gly 115 120 125 Val ArgAsn Gly Glu Ala Leu Ala Val Asn Phe Pro Leu Val Leu His 130 135 140 HisMet Pro Asp Glu Ser Val Thr Val Glu Asn His Arg Asp Arg Leu 145 150 155160 Leu Arg Leu Val Lys His Leu Ser Pro Asn Val Val Thr Leu Val Glu 165170 175 Gln Glu Ala Asn Thr Asn Thr Ala Pro Phe Leu Pro Arg Phe Val Glu180 185 190 Thr Met Asn His Tyr Leu Ala Val Phe Glu Ser Ile Asp Val LysLeu 195 200 205 Ala Arg Asp His Lys Glu Arg Ile Asn Val Glu Gln His CysLeu Ala 210 215 220 Arg Glu Val Glu Asn Leu Ile Ala Cys Glu Gly Val GluArg Glu Glu 225 230 235 240 Arg His Glu Pro Leu Gly Lys Trp Arg Ser ArgPhe His Met Ala Gly 245 250 255 Phe Lys Pro Tyr Pro Leu Ser Ser Tyr ValAsn Ala Thr Ile Lys Gly 260 265 270 Leu Leu Glu Ser Tyr Ser Glu Lys TyrThr Leu Glu Glu Arg Asp Gly 275 280 285 Ala Leu Tyr Leu Gly Trp Lys AsnGln Pro Leu Ile Thr Ser Cys Ala 290 295 300 Trp Arg 305 20 1231 DNAArabidopsis thaliana 20 gctatggaag gagagaagat ggttcatgtg attgatctcgatgcttctga gccagctcaa 60 tggcttgctt tgcttcaagc ttttaactct aggcctgaaggtccacctca tttgagaatc 120 actggtgttc atcaccagaa ggaagtgctt gaacaaatggctcatagact cattgaggaa 180 gcagagaaac tcgatatccc gtttcagttt aatcccgttgtgagtaggtt agactgttta 240 aatgtagaac agttgcgggt taaaacagga gaggccttagccgttagctc ggttcttcaa 300 ttgcatacct tcttggcctc tgatgatgat ctcatgagaaagaactgcgc tttacggttt 360 cagaacaacc ctagtggagt tgacttgcag agagttctaatgatgagcca tggctctgca 420 gctgaggcac gtgagaatga tatgagtaac aacaatgggtatagccctag cggtgactcg 480 gcctcatctt tgcctttacc aagttcagga aggactgatagcttcctcaa tgctatttgg 540 ggtttgtctc caaaggtcat ggtggtcact gagcaagactcagaccacaa cggctccaca 600 ctaatggaga ggctattaga atcactttac acctacgcagcattgtttga ttgcttggaa 660 acaaaagttc caagaacgtc tcaagatagg atcaaagtggagaagatgct cttcggggag 720 gagatcaaga acatcatatc ctgcgaggga tttgagagaagagaaagaca cgagaagctt 780 gagaaatgga gccagaggat cgatttggct ggttttgggaatgttcctct tagctattat 840 gcgatgttgc aggctaggag attgcttcaa gggtgcggttttgatgggta tagaatcaag 900 gaagagagcg ggtgcgcagt aatttgctgg caagatcgacctctatactc ggtatcagct 960 tggagatgca ggaagtgaat gatatattac agtttgtcttctattttggt tatgagcaga 1020 gtccctttct tttttgtata catggggaca caatcttagttgttttgtga tggtgacttt 1080 ctgtctcttt atgctatttt ggcttaaatg cttctactgcctctgcatgt aaagcctttg 1140 tgtgttggtt caatttggtc tggtgtgggt gtaataccaaaccaaatcca atttgagctg 1200 aagataacta atttgatgat cggctcgtgc c 1231 21325 PRT Arabidopsis thaliana 21 Ala Met Glu Gly Glu Lys Met Val His ValIle Asp Leu Asp Ala Ser 1 5 10 15 Glu Pro Ala Gln Trp Leu Ala Leu LeuGln Ala Phe Asn Ser Arg Pro 20 25 30 Glu Gly Pro Pro His Leu Arg Ile ThrGly Val His His Gln Lys Glu 35 40 45 Val Leu Glu Gln Met Ala His Arg LeuIle Glu Glu Ala Glu Lys Leu 50 55 60 Asp Ile Pro Phe Gln Phe Asn Pro ValVal Ser Arg Leu Asp Cys Leu 65 70 75 80 Asn Val Glu Gln Leu Arg Val LysThr Gly Glu Ala Leu Ala Val Ser 85 90 95 Ser Val Leu Gln Leu His Thr PheLeu Ala Ser Asp Asp Asp Leu Met 100 105 110 Arg Lys Asn Cys Ala Leu ArgPhe His Asn Asn Pro Ser Gly Val Asp 115 120 125 Leu Gln Arg Val Leu MetMet Ser His Gly Ser Ala Ala Glu Ala Arg 130 135 140 Glu Asn Asp Met SerAsn Asn Asn Gly Tyr Ser Pro Ser Gly Asp Ser 145 150 155 160 Ala Ser SerLeu Pro Leu Pro Ser Ser Gly Arg Thr Asp Ser Phe Leu 165 170 175 Asn AlaIle Trp Gly Leu Ser Pro Lys Val Met Val Val Thr Glu Gln 180 185 190 AspSer Asp His Asn Gly Ser Thr Leu Met Glu Arg Leu Leu Glu Ser 195 200 205Leu Tyr Thr Tyr Ala Ala Leu Phe Asp Cys Leu Glu Thr Lys Val Pro 210 215220 Arg Thr Ser Gln Asp Arg Ile Lys Val Glu Lys Met Leu Phe Gly Glu 225230 235 240 Glu Ile Lys Asn Ile Ile Ser Cys Glu Gly Phe Glu Arg Arg GluArg 245 250 255 His Glu Lys Leu Glu Lys Trp Ser Gln Arg Ile Asp Leu AlaGly Phe 260 265 270 Gly Asn Val Pro Leu Ser Tyr Tyr Ala Met Leu Gln AlaArg Arg Leu 275 280 285 Leu Gln Gly Cys Gly Phe Asp Gly Tyr Arg Ile LysGlu Glu Ser Gly 290 295 300 Cys Ala Val Ile Cys Trp Gln Asp Arg Pro LeuTyr Ser Val Ser Ala 305 310 315 320 Trp Arg Cys Arg Lys 325 22 1368 DNAArabidopsis thaliana 22 ctttgtcaat ggtaaatgag ctgaggcaga tagtttctatccaaggagac ccttctcaga 60 gaatcgcagc ttacatggtg gaaggtctag ctgcaagaatggccgcttca ggaaaattca 120 tctacagagc attgaaatgc aaagagcctc cttcggatgagaggcttgca gctatgcaag 180 tcctgtttga agtctgccct tgtttcaagt tcgggtttttagcagctaat ggtgcgatac 240 ttgaagcaat caaaggtgaa gaagaagttc acataatcgatttcgatata aaccaaggga 300 accaatacat gacactgata cgaagcattg ctgagttgcctggtaaacga cctcgcctga 360 ggttaacagg aattgatgac cctgaatcag tccaacgctccattggaggg ctaagaatca 420 tcggtctaag actcgagcaa ctcgcagagg ataatggagtatccttcaaa ttcaaagcaa 480 tgccttcaaa gacttcgatt gtctctccat caacactcggttgcaaacca ggagaaacct 540 taatagtgaa ctttgcattc caacttcacc acatgcctgacgagagtgtc acaacagtaa 600 accagcggga cgagctactt cacatggtca aaagcttaaacccaaagctt gtcacggtcg 660 ttgaacaaga cgtgaacaca aacacttcac cgttctttcccagattcata gaggcttacg 720 aatactactc agcagttttc gagtctctag acatgacacttccaagagaa agccaagaga 780 ggatgaatgt agaaagacag tgtctcgcta gagacatagtcaacattgtt gcttgcgaag 840 gagaagaacg gatagagaga tacgaggctg cgggaaaatggagagcaagg atgatgatgg 900 ctggattcaa tccaaaacca atgagtgcta aagtaaccaacaatatacaa aacctgataa 960 agcaacaata ttgcaataag tacaagctta aagaagaaatgggtgagctc catttttgct 1020 gggaggagaa aagcttaatc gttgcttcag cttggaggtaagataagtga caagagcata 1080 tagtctttat gtttcataaa acataattat gtttttactgtaatcttggg ttattgtgta 1140 actggttaaa tcatctccat gtattattac cagaggttaggggtgatcac aggtactaaa 1200 agctaatcta acacttatgg aagaattttt ctttcttttttttccctatt atataaaaat 1260 aattagagtt ttggttctaa acctatttgc taagtgtgaatgagtcttta catgttcata 1320 tttcagttca aatggttaaa tttgttaagg ttctcacttaaaaaaaaa 1368 23 351 PRT Arabidopsis thaliana 23 Leu Ser Met Val Asn GluLeu Arg Gln Ile Val Ser Ile Gln Gly Asp 1 5 10 15 Pro Ser Gln Arg IleAla Ala Tyr Met Val Glu Gly Leu Ala Ala Arg 20 25 30 Met Ala Ala Ser GlyLys Phe Ile Tyr Arg Ala Leu Lys Cys Lys Glu 35 40 45 Pro Pro Ser Asp GluArg Leu Ala Ala Met Gln Val Leu Phe Glu Val 50 55 60 Cys Pro Cys Phe LysPhe Gly Phe Leu Ala Ala Asn Gly Ala Ile Leu 65 70 75 80 Glu Ala Ile LysGly Glu Glu Glu Val His Ile Ile Asp Phe Asp Ile 85 90 95 Asn Gln Gly AsnGln Tyr Met Thr Leu Ile Arg Ser Ile Ala Glu Leu 100 105 110 Pro Gly LysArg Pro Arg Leu Arg Leu Thr Gly Ile Asp Asp Pro Glu 115 120 125 Ser ValGln Arg Ser Ile Gly Gly Leu Arg Ile Ile Asn Leu Arg Leu 130 135 140 GluGln Leu Ala Glu Asp Asn Gly Val Ser Phe Lys Phe Lys Ala Met 145 150 155160 Pro Ser Lys Thr Ser Ile Val Ser Pro Ser Thr Leu Gly Cys Lys Pro 165170 175 Gly Glu Thr Leu Ile Val Asn Phe Ala Phe Gln Leu His His Met Pro180 185 190 Asp Glu Ser Val Thr Thr Val Asn Gln Arg Asp Glu Leu Leu HisMet 195 200 205 Val Lys Ser Leu Asn Pro Leu Val Thr Val Val Glu Gln AspVal Asn 210 215 220 Thr Asn Thr Ser Pro Phe Phe Pro Arg Phe Ile Glu AlaTyr Glu Tyr 225 230 235 240 Tyr Ser Ala Val Phe Glu Ser Leu Asp Met ThrLeu Pro Arg Glu Ser 245 250 255 Gln Glu Arg Met Asn Val Glu Arg Gln CysLeu Ala Arg Asp Ile Val 260 265 270 Asn Ile Val Ala Cys Glu Gly Glu GluArg Ile Glu Arg Tyr Glu Ala 275 280 285 Ala Gly Lys Trp Arg Ala Arg MetMet Met Ala Gly Phe Asn Pro Lys 290 295 300 Pro Met Ser Ala Lys Val ThrAsn Asn Ile Gln Asn Leu Ile Lys Gln 305 310 315 320 Gln Tyr Cys Asn LysTyr Lys Leu Lys Glu Glu Met Gly Glu Leu His 325 330 335 Phe Cys Trp GluGlu Lys Ser Leu Ile Val Ala Ser Ala Trp Arg 340 345 350 24 100 DNA Zeamays 24 ccaggaggcg ttcgagcggg aggagcgtgt gcacatcatc gacctcgacatcatgcaggg 60 gctgcagtgg ccgggcctcc tccacatcct tgcctcccgc 100 25 33 PRTZea mays 25 Gln Glu Ala Phe Glu Arg Glu Glu Arg Val His Ile Ile Asp LeuAsp 1 5 10 15 Ile Met Gln Gly Leu Gln Trp Pro Gly Leu Phe His Ile LeuAla Ser 20 25 30 Arg 26 1094 DNA Zea mays 26 ccacgcgtcc gtcaaaggatacaaccatgt acacataatt gacttttccc tgatgcaagg 60 tctccagtgg ccggcactcatggatgtctt ctccgcccgt gagggtgggc caccaaagct 120 ccgaatcaca ggcattggcccgaacccaat aggtggccgt gacgagctcc atgaagtggg 180 aattcgcctc gccaagtatgcacactcggt gggtatcgac ttcactttcc agggagtctg 240 tgtcgatcaa cttgataggttgtgcgactg gatgcttctc aaaccaatca aaggagaggc 300 agttgccata aactccatcctacaactcca tcgcctcctc gttgacccag atgcaaaccc 360 agtggtgccc gcaccaatagatatcctcct caaattggtc atcaagataa accccatgat 420 cttcacggtg gttgagcatgaggcagatca caacagacca ccactactag agaggttcac 480 taatgccctc ttccactatgcgaccatgtt tgactctttg gaggccatgc atcgttgtac 540 cagtggtaga gacatcaccgactcactcac agaggtgtac cttcgaggtg agatttttga 600 cattgtctgc ggcgagggcagtgcacgcac cgaacgtcat gagttgtttg gtcactggag 660 ggagaggctc acctatgctgggctaactca agtgtggttc gaccccgatg aggttgacac 720 gctaaaagac cagttgatccatgtgacatc cttatctggc tctgggttca acatcctagt 780 gtgtgatggc agccttgcactagcgtggca taatcgcccg ttatatgtgg caacagcttg 840 gtgtgtgaca ggaggaaatgctgccagttc catggttggc aacatctgta agggtacaaa 900 tgatagtaga agaaaggaaaaccgtaatgg acccatggag tagcaggaag aataaccatg 960 tcatgagcaa atcgatcaagtaataaaatg cactgatgac atgcatggtg atctaaagtt 1020 tttttgcgtg aatgtgcaatgacgaattgt tcaatttgaa taacctaatc atgagactca 1080 aaaaaaaaaa aaaa 1094 27313 PRT Arabidopsis thaliana 27 His Ala Ser Val Lys Gly Tyr Asn His ValHis Ile Ile Asp Phe Ser 1 5 10 15 Leu Met Gln Gly Leu Gln Trp Pro AlaLeu Met Asp Val Phe Ser Ala 20 25 30 Arg Glu Gly Gly Pro Pro Lys Leu ArgIle Thr Gly Ile Gly Pro Asn 35 40 45 Pro Ile Gly Gly Arg Asp Glu Leu HisGlu Val Gly Ile Arg Leu Ala 50 55 60 Lys Tyr Ala His Ser Val Gly Ile AspPhe Thr Phe Gln Gly Val Cys 65 70 75 80 Val Asp Gln Leu Asp Arg Leu CysAsp Trp Met Leu Leu Lys Pro Ile 85 90 95 Lys Gly Glu Ala Val Ala Ile AsnSer Ile Leu Gln Leu His Arg Leu 100 105 110 Leu Val Asp Pro Asp Ala AsnPro Val Val Pro Ala Pro Ile Asp Ile 115 120 125 Leu Leu Lys Leu Val IleLys Ile Asn Pro Met Ile Phe Thr Val Val 130 135 140 Glu His Glu Ala AspHis Asn Arg Pro Pro Leu Leu Glu Arg Phe Thr 145 150 155 160 Asn Ala LeuPhe His Tyr Ala Thr Met Phe Asp Ser Leu Glu Ala Met 165 170 175 His ArgCys Thr Ser Gly Arg Asp Ile Thr Asp Ser Leu Thr Glu Val 180 185 190 TyrLeu Arg Gly Glu Ile Phe Asp Ile Val Cys Gly Glu Gly Ser Ala 195 200 205Arg Thr Glu Arg His Glu Leu Phe Gly His Trp Arg Glu Arg Leu Thr 210 215220 Tyr Ala Gly Leu Thr Gln Val Trp Phe Asp Pro Asp Glu Val Asp Thr 225230 235 240 Leu Lys Asp Gln Leu Ile His Val Thr Ser Leu Ser Gly Ser GlyPhe 245 250 255 Asn Ile Leu Val Cys Asp Gly Ser Leu Ala Leu Ala Trp HisAsn Arg 260 265 270 Pro Leu Tyr Val Ala Thr Ala Trp Cys Val Thr Gly GlyAsn Ala Ala 275 280 285 Ser Ser Met Val Gly Asn Ile Cys Lys Gly Thr AsnAsp Ser Arg Arg 290 295 300 Lys Glu Asn Arg Asn Gly Pro Met Glu 305 31028 611 DNA Oryza sp. 28 cccaacttgg gaagcccttc ctccgctccg cctcctacctcaaggaggcc ctcctcctcg 60 cactcgccga cagccaccat ggctcctccg gcgtcacctcgccgctcgac gttgccctca 120 agcttgcagc atacaagtct ttctctgacc tgtcacctgtgctccagttc actaacttta 180 ccgcaacaag gcgcttcttg atgagattgg tggcatggcaacttcctgca tccatgtcat 240 tgactttgat ctcggtgttg gtggtcagtg ggcttccttcttgcaggagc ttgcccaccg 300 ccggggagct ggaggtatgg ccttgccgtt gttgaagctcacggctttca tgtcgactgc 360 ttctcaccat ccactggagc tgcaccttac ccaggataacctctctcagt ttgccgcaga 420 gctcagaatt cctttcgaat tcaatgccgt cagtcttgatgcattcaatc ctgcggaatc 480 tatttcttcc tctggtgatg aagttgttgc tgttagcctccctgttggct gctctgctcg 540 tgcaccaccg ctgccagcga ttcttcggtt ggtgaaacagctttgtccta aggttgtcgt 600 ggctattgat c 611 29 502 DNA Oryza sp. 29tttttttttt tttttttttt tttttttttt tacagagcaa cagcagtata atattaattc 60tgtaccacac aaccatttga taggttaaat taccctctag tctctactca taagcagtgt 120ttccaatgag atgatcatgg ctaattgagc agagcatggc aacaacctaa agcaacatca 180ttagctatag agactgacac caatattcct aaatccacta ggctagctaa taagctgcaa 240cgaaaagcaa tatgaagagt tcaacagctc aagacaacaa tttcatttgc aacatttaat 300tgcaagaata aatggacatt actggagtgg tcgatgcttg caaacggtgg tggaaccttg 360gtggagtgaa gcttatggct gatcagcacc gccaagatga tatggataca agctccccac 420gctgccagta gagcgtaaga gcagctccgc gtttctccac atggaatcct cggacctgca 480cccgcttcag gaggcagtct gc 502 30 298 PRT Arabidopsis thaliana 30 Pro GlnGln Gln Gln Gln His Gln Gln Gln Gln Gln Gln His Lys Pro 1 5 10 15 ProPro Pro Pro Ile Gln Gln Gln Glu Arg Glu Asn Ser Ser Thr Asp 20 25 30 AlaPro Pro Gln Pro Glu Thr Val Thr Ala Thr Val Pro Ala Val Gln 35 40 45 ThrAsn Thr Ala Glu Ala Leu Arg Glu Arg Lys Glu Glu Ile Lys Arg 50 55 60 GlnLys Gln Asp Glu Glu Gly Leu His Leu Leu Thr Leu Leu Leu Gln 65 70 75 80Cys Ala Glu Ala Val Ser Ala Asp Asn Leu Glu Glu Ala Asn Lys Leu 85 90 95Leu Leu Glu Ile Ser Gln Leu Ser Thr Pro Tyr Gly Thr Ser Ala Gln 100 105110 Arg Val Ala Ala Tyr Phe Ser Glu Ala Met Ser Ala Arg Leu Leu Asn 115120 125 Ser Cys Leu Gly Ile Tyr Ala Ala Leu Pro Ser Arg Trp Met Pro Gln130 135 140 Thr His Ser Leu Lys Met Val Ser Ala Phe Gln Val Phe Asn GlyIle 145 150 155 160 Ser Pro Leu Val Lys Phe Ser His Phe Thr Ala Asn GlnAla Ile Gln 165 170 175 Glu Ala Phe Glu Lys Glu Asp Ser Val His Ile IleAsp Leu Asp Ile 180 185 190 Met Gln Gly Leu Gln Trp Pro Gly Leu Phe HisIle Leu Ala Ser Arg 195 200 205 Pro Gly Gly Pro Pro His Val Arg Leu ThrGly Leu Gly Thr Ser Met 210 215 220 Glu Ala Leu Gln Ala Thr Gly Lys ArgLeu Ser Asp Phe Thr Asp Lys 225 230 235 240 Leu Gly Leu Pro Phe Glu PheCys Pro Leu Ala Glu Lys Val Gly Asn 245 250 255 Asp Leu Thr Glu Arg LeuAsn Val Arg Lys Arg Glu Ala Ala Val His 260 265 270 Trp Leu Gln His SerLeu Tyr Asp Val Thr Gly Ser Asp Ala His Thr 275 280 285 Leu Trp Leu LeuGln Arg Leu Ala Pro Lys 290 295 31 307 PRT Arabidopsis thaliana VARIANT(1)...(307) Xaa = Any Amino Acid 31 Gly Thr Ser Pro Thr Gly Pro Glu LeuLeu Thr Tyr Met His Ile Leu 1 5 10 15 Tyr Glu Ala Cys Pro Tyr Phe LysPhe Gly Tyr Glu Ser Ala Asn Gly 20 25 30 Ala Ile Ala Glu Ala Val Lys AsnGlu Ser Phe Val His Ile Ile Asp 35 40 45 Phe Gln Ile Ser Gln Gly Gly GlnTrp Val Ser Leu Ile Arg Ala Leu 50 55 60 Gly Ala Arg Pro Gly Gly Pro ProAsn Val Arg Ile Thr Gly Ile Asp 65 70 75 80 Asp Pro Arg Ser Ser Phe AlaArg Gln Gly Gly Leu Glu Leu Val Gly 85 90 95 Gln Arg Leu Gly Lys Leu AlaGlu Met Cys Gly Val Pro Phe Glu Phe 100 105 110 His Gly Ala Ala Leu CysCys Thr Glu Val Glu Ile Glu Lys Leu Gly 115 120 125 Val Arg Asn Gly GluAla Leu Ala Val Asn Phe Pro Leu Val Leu His 130 135 140 His Met Pro AspGlu Ser Val Thr Val Glu Asn His Arg Asp Arg Leu 145 150 155 160 Leu ArgLeu Val Lys His Leu Ser Pro Asn Val Val Thr Leu Val Glu 165 170 175 GlnGlu Ala Asn Thr Asn Thr Ala Pro Phe Leu Pro Arg Phe Val Glu 180 185 190Thr Met Asn His Tyr Leu Ala Val Phe Glu Ser Ile Asp Val Lys Leu 195 200205 Ala Arg Asp His Lys Glu Arg Ile Asn Val Glu Gln His Cys Leu Ala 210215 220 Arg Glu Val Val Asn Leu Ile Ala Cys Glu Gly Val Glu Arg Glu Glu225 230 235 240 Arg His Glu Pro Leu Gly Lys Trp Arg Ser Arg Phe His MetAla Gly 245 250 255 Phe Lys Pro Tyr Pro Leu Ser Ser Tyr Val Asn Ala ThrIle Lys Gly 260 265 270 Leu Leu Glu Ser Tyr Ser Glu Lys Tyr Thr Leu GluGlu Arg Asp Gly 275 280 285 Ala Leu Tyr Leu Gly Trp Lys Asn Gln Pro LeuIle Thr Ser Cys Ala 290 295 300 Trp Arg Xaa 305 32 353 PRT Arabidopsisthaliana VARIANT (1)...(353) Xaa = Any Amino Acid 32 Leu Ser Met Val AsnGlu Leu Arg Gln Ile Val Ser Ile Gln Gly Asp 1 5 10 15 Pro Ser Gln ArgIle Ala Ala Tyr Met Val Glu Gly Leu Ala Ala Arg 20 25 30 Met Ala Ala SerGly Lys Phe Ile Tyr Arg Ala Leu Lys Cys Lys Glu 35 40 45 Pro Pro Ser AspGlu Arg Leu Ala Ala Met Gln Val Leu Phe Glu Val 50 55 60 Cys Pro Cys PheLys Phe Gly Phe Leu Ala Ala Asn Gly Ala Ile Leu 65 70 75 80 Glu Ala IleLys Gly Glu Glu Glu Val His Ile Ile Asp Phe Asp Ile 85 90 95 Asn Gln GlyAsn Gln Tyr Met Thr Leu Ile Arg Ser Ile Ala Glu Leu 100 105 110 Pro GlyLys Arg Pro Arg Leu Arg Leu Thr Gly Ile Asp Asp Pro Glu 115 120 125 SerVal Gln Arg Ser Ile Gly Gly Leu Arg Ile Ile Gly Leu Arg Leu 130 135 140Glu Gln Leu Ala Glu Asp Asn Gly Val Ser Phe Lys Phe Lys Ala Met 145 150155 160 Pro Ser Lys Thr Ser Ile Val Ser Pro Ser Thr Leu Gly Cys Lys Pro165 170 175 Gly Glu Thr Leu Ile Val Asn Phe Ala Phe Gln Leu His His MetPro 180 185 190 Asp Glu Ser Val Thr Thr Val Asn Gln Arg Asp Glu Leu LeuHis Met 195 200 205 Val Lys Ser Leu Asn Pro Lys Leu Val Thr Val Val GluGln Asp Val 210 215 220 Asn Thr Asn Thr Ser Pro Phe Phe Pro Arg Phe IleGlu Ala Tyr Glu 225 230 235 240 Tyr Tyr Ser Ala Val Phe Glu Ser Leu AspMet Thr Leu Pro Arg Glu 245 250 255 Ser Gln Glu Arg Met Asn Val Glu ArgGln Cys Leu Ala Arg Asp Ile 260 265 270 Val Asn Ile Val Ala Cys Glu GlyGlu Glu Arg Ile Glu Arg Tyr Glu 275 280 285 Ala Ala Gly Lys Trp Arg AlaArg Met Met Met Ala Gly Phe Asn Pro 290 295 300 Lys Pro Met Ser Ala LysVal Thr Asn Asn Ile Gln Asn Leu Ile Lys 305 310 315 320 Gln Gln Tyr CysAsn Lys Tyr Lys Leu Lys Glu Glu Met Gly Glu Leu 325 330 335 His Phe CysTrp Glu Glu Lys Ser Leu Ile Val Ala Ser Ala Trp Arg 340 345 350 Xaa 33326 PRT Arabidopsis thaliana VARIANT (1)...(326) Xaa = Any Amino Acid 33Ala Met Glu Gly Glu Lys Met Val His Val Ile Asp Leu Asp Ala Ser 1 5 1015 Glu Pro Ala Gln Trp Leu Ala Leu Leu Gln Ala Phe Asn Ser Arg Pro 20 2530 Glu Gly Pro Pro His Leu Arg Ile Thr Gly Val His His Gln Lys Glu 35 4045 Val Leu Glu Gln Met Ala His Arg Leu Ile Glu Glu Ala Glu Lys Leu 50 5560 Asp Ile Pro Phe Gln Phe Asn Pro Val Val Ser Arg Leu Asp Cys Leu 65 7075 80 Asn Val Glu Gln Leu Arg Val Lys Thr Gly Glu Ala Leu Ala Val Ser 8590 95 Ser Val Leu Gln Leu His Thr Phe Leu Ala Ser Asp Asp Asp Leu Met100 105 110 Arg Lys Asn Cys Ala Leu Arg Phe Gln Asn Asn Pro Ser Gly ValAsp 115 120 125 Leu Gln Arg Val Leu Met Met Ser His Gly Ser Ala Ala GluAla Arg 130 135 140 Glu Asn Asp Met Ser Asn Asn Asn Gly Tyr Ser Pro SerGly Asp Ser 145 150 155 160 Ala Ser Ser Leu Pro Leu Pro Ser Ser Gly ArgThr Asp Ser Phe Leu 165 170 175 Asn Ala Ile Trp Gly Leu Ser Pro Lys ValMet Val Val Thr Glu Gln 180 185 190 Asp Ser Asp His Asn Gly Ser Thr LeuMet Glu Arg Leu Leu Glu Ser 195 200 205 Leu Tyr Thr Tyr Ala Ala Leu PheAsp Cys Leu Glu Thr Lys Val Pro 210 215 220 Arg Thr Ser Gln Asp Arg IleLys Val Glu Lys Met Leu Phe Gly Glu 225 230 235 240 Glu Ile Lys Asn IleIle Ser Cys Glu Gly Phe Glu Arg Arg Glu Arg 245 250 255 His Glu Lys LeuGlu Lys Trp Ser Gln Arg Ile Asp Leu Ala Gly Phe 260 265 270 Gly Asn ValPro Leu Ser Tyr Tyr Ala Met Leu Gln Ala Arg Arg Leu 275 280 285 Leu GlnGly Cys Gly Phe Asp Gly Tyr Arg Ile Lys Glu Glu Ser Gly 290 295 300 CysAla Val Ile Cys Trp Gln Asp Arg Pro Leu Tyr Ser Val Ser Ala 305 310 315320 Trp Arg Cys Arg Lys Xaa 325 34 277 PRT Arabidopsis thaliana VARIANT(1)...(277) Xaa = Any Amino Acid 34 Asn Lys Arg Leu Lys Ser Cys Ser SerPro Asp Ser Met Val Thr Ser 1 5 10 15 Thr Ser Thr Gly Thr Gln Ile GlyGly Val Ile Gly Thr Thr Val Thr 20 25 30 Thr Thr Thr Thr Thr Thr Thr AlaAla Ala Glu Ser Thr Arg Ser Val 35 40 45 Ile Leu Val Asp Ser Gln Glu AsnGly Val Arg Leu Val His Ala Leu 50 55 60 Met Ala Cys Ala Glu Ala Ile GlnGln Asn Asn Leu Thr Leu Ala Glu 65 70 75 80 Ala Leu Val Lys Gln Ile GlyCys Leu Ala Val Ser Gln Ala Gly Ala 85 90 95 Met Arg Lys Val Ala Thr TyrPhe Ala Glu Ala Leu Ala Arg Arg Ile 100 105 110 Tyr Arg Leu Ser Pro ProGln Asn Gln Ile Asp His Cys Leu Ser Asp 115 120 125 Thr Leu Gln Met HisPhe Tyr Glu Thr Cys Pro Tyr Leu Lys Phe Ala 130 135 140 His Phe Thr AlaAsn Gln Ala Ile Leu Glu Ala Phe Glu Gly Lys Lys 145 150 155 160 Arg ValHis Val Ile Asp Phe Ser Met Asn Gln Gly Leu Gln Trp Pro 165 170 175 AlaLeu Met Gln Ala Leu Ala Leu Arg Glu Gly Gly Pro Pro Thr Phe 180 185 190Arg Leu Thr Gly Ile Gly Pro Pro Ala Pro Asp Asn Ser Asp His Leu 195 200205 His Glu Val Gly Cys Lys Leu Ala Gln Leu Ala Glu Ala Ile His Val 210215 220 Glu Phe Glu Tyr Arg Gly Phe Val Ala Asn Ser Leu Ala Asp Leu Asp225 230 235 240 Ala Ser Met Leu Glu Leu Arg Pro Ser Asp Thr Glu Ala ValAla Val 245 250 255 Asn Ser Val Phe Glu Leu His Lys Leu Leu Gly Arg XaaGly Gly Ile 260 265 270 Glu Lys Val Leu Gly 275 35 262 PRT Arabidopsisthaliana 35 Gly Gly Gly Gly Asp Thr Tyr Thr Thr Asn Lys Arg Leu Lys CysSer 1 5 10 15 Asn Gly Val Val Glu Thr Thr Thr Ala Thr Ala Glu Ser ThrArg His 20 25 30 Val Val Leu Val Asp Ser Gln Glu Asn Gly Val Arg Leu ValHis Ala 35 40 45 Leu Leu Ala Cys Ala Glu Ala Val Gln Lys Glu Asn Leu ThrVal Ala 50 55 60 Glu Ala Leu Val Lys Gln Ile Gly Phe Leu Ala Val Ser GlnIle Gly 65 70 75 80 Ala Met Arg Gln Val Ala Thr Tyr Phe Ala Glu Ala LeuAla Arg Arg 85 90 95 Ile Tyr Arg Leu Ser Pro Ser Gln Ser Pro Ile Asp HisSer Leu Ser 100 105 110 Asp Thr Leu Gln Met His Phe Tyr Glu Thr Cys ProTyr Leu Lys Phe 115 120 125 Ala His Phe Thr Ala Asn Gln Ala Ile Leu GluAla Phe Gln Gly Lys 130 135 140 Lys Arg Val His Val Ile Asp Phe Ser MetSer Gln Gly Leu Gln Trp 145 150 155 160 Pro Ala Leu Met Gln Ala Leu AlaLeu Arg Pro Gly Gly Pro Pro Val 165 170 175 Phe Arg Leu Thr Gly Ile GlyPro Pro Ala Pro Asp Asn Phe Asp Tyr 180 185 190 Leu His Glu Val Gly CysLys Leu Ala His Leu Ala Glu Ala Ile His 195 200 205 Val Glu Phe Glu TyrArg Gly Phe Val Ala Asn Thr Leu Ala Asp Leu 210 215 220 Asp Ala Ser MetLeu Glu Leu Arg Pro Ser Glu Ile Glu Ser Val Ala 225 230 235 240 Val AsnSer Val Phe Glu Leu His Lys Leu Leu Gly Arg Pro Gly Ala 245 250 255 IleAsp Lys Val Leu Gly 260 36 203 PRT Oryza sp. 36 Gln Leu Gly Lys Pro PheLeu Arg Ser Ala Ser Tyr Leu Lys Glu Ala 1 5 10 15 Leu Leu Leu Ala LeuAla Asp Ser His His Gly Ser Ser Gly Val Thr 20 25 30 Ser Pro Leu Asp ValAla Leu Lys Leu Ala Ala Tyr Lys Ser Phe Ser 35 40 45 Asp Leu Ser Pro ValLeu Gln Phe Thr Asn Phe Thr Ala Asn Lys Ala 50 55 60 Leu Leu Asp Glu IleGly Gly Met Ala Thr Ser Cys Ile His Val Ile 65 70 75 80 Asp Phe Asn LeuGly Val Gly Gly Gln Trp Ala Ser Phe Leu Gln Glu 85 90 95 Leu Ala His ArgArg Gly Ala Gly Gly Met Ala Leu Pro Leu Leu Lys 100 105 110 Leu Thr AlaPhe Met Ser Thr Ala Ser His His Pro Leu Glu Leu His 115 120 125 Leu ThrGln Asp Asn Leu Ser Gln Phe Ala Ala Glu Leu Arg Ile Pro 130 135 140 PheGlu Phe Asn Ala Val Ser Leu Asp Ala Phe Asn Pro Ala Glu Ser 145 150 155160 Ile Ser Ser Ser Gly Asp Glu Val Val Ala Val Ser Leu Pro Val Gly 165170 175 Cys Ser Ala Arg Ala Pro Pro Leu Pro Ala Ile Leu Arg Leu Val Lys180 185 190 Gln Leu Cys Pro Lys Val Val Val Ala Ile Asp 195 200 37 131PRT Zea mays 37 His Ala Ser Val Lys Gly Tyr Asn His Val His Ile Ile AspPhe Ser 1 5 10 15 Leu Met Gln Gly Leu Gln Trp Pro Ala Leu Met Asp ValPhe Ser Ala 20 25 30 Arg Glu Gly Gly Pro Pro Lys Leu Arg Ile Thr Gly IleGly Pro Asn 35 40 45 Pro Ile Gly Gly Arg Asp Glu Leu His Glu Val Gly IleArg Leu Ala 50 55 60 Lys Tyr Ala His Ser Val Gly Ile Asp Phe Thr Phe GlnGly Val Cys 65 70 75 80 Val Asp Gln Leu Asp Arg Leu Cys Asp Trp Met LeuLeu Lys Pro Ile 85 90 95 Lys Gly Glu Ala Val Ala Ile Asn Ser Ile Leu GlnLeu His Arg Leu 100 105 110 Leu Val Asp Pro Asp Ala Asn Pro Val Val ProAla Pro Ile Asp Ile 115 120 125 Leu Leu Lys 130 38 33 PRT Arabidopsisthaliana 38 Gln Glu Ala Phe Glu Arg Glu Glu Arg Val His Ile Ile Asp LeuAsp 1 5 10 15 Ile Met Gln Gly Leu Gln Trp Pro Gly Leu Phe His Ile LeuAla Ser 20 25 30 Arg 39 29 PRT Arabidopsis thaliana VARIANT (1)...(29)Xaa = Any Amino Acid 39 Phe Ala Gly Cys Arg Arg Val His Val Val Asp PheGly Ile Lys Gln 1 5 10 15 Gly Met Gln Trp Pro Ala Leu Leu Xaa Asp LeuAla Leu 20 25 40 73 PRT Homo sapiens VARIANT (1)...(73) Xaa = Any AminoAcid 40 Gly Arg Asn Gly Arg Thr Leu Trp Leu Gly Glu Gly His Ile Asp Leu1 5 10 15 Trp Pro Leu Gln Gly Leu Leu Ser Gln Gly Leu Gln Arg Ala LeuCys 20 25 30 Ala Arg Pro Leu Gly Ala Pro His Val Phe Leu Pro Gly Leu HisThr 35 40 45 Leu Ser Leu Gly Leu Gln Xaa Arg His Leu Leu Val His Met MetAla 50 55 60 Leu Ser Tyr Ser Tyr Gly Arg Xaa Pro 65 70 41 59 PRTArabidopsis thaliana 41 Thr Ser Asp Ser Ala Ser Ser Phe Asn Ile Pro ThrSer Ala Gln Asn 1 5 10 15 His Tyr Ala Thr Gly Ser Phe Ser Thr Asn SerArg Thr Thr Asn Val 20 25 30 Ala Thr Ala Thr Thr Asn Ser Ala Thr Ala HisTrp Val Ala Thr Asp 35 40 45 Ala Glu His Thr Asp Thr Ile Ile Ala Gln Pro50 55 42 110 PRT Brassica sp. VARIANT (1)...(110) Xaa = Any Amino Acid42 Arg Xaa Phe Asp Ser Leu Glu His Asp Ala Ser Lys Gly Glu Pro Arg 1 510 15 Glu Asp Glu Arg Gly Arg Xaa Cys Leu Ala Arg Asn Ile Val Asn Ile 2025 30 Val Xaa Cys Lys Xaa Glu Glu Arg Ile Glu Arg Tyr Glu Val Thr Gly 3540 45 Lys Trp Arg Ala Arg Met Met Met Ala Gly Phe Ser Pro Arg Pro Met 5055 60 Ser Gly Arg Val Thr Ser Asn Ile Glu Ser Leu Ile Lys Arg Asp Tyr 6570 75 80 Cys Ser Lys Tyr Lys Val Lys Glu Glu Met Gly Glu Leu His Phe Ser85 90 95 Trp Glu Glu Lys Ser Leu Ile Val Ala Ser Ala Trp Ser Xaa 100 105110 43 137 PRT Oryza sp. VARIANT (1)...(137) Xaa = Any Amino Acid 43 AsnGly Ser Tyr Asn Ala Pro Phe Phe Val Thr Arg Phe Arg Glu Ala 1 5 10 15Leu Phe His Tyr Ser Ala Ile Phe Asp Met Leu Glu Thr Asn Ile Pro 20 25 30Lys Asp Asn Glu Gln Arg Leu Leu Ile Glu Ser Ala Leu Phe Ser Arg 35 40 45Glu Xaa Asn Val Ile Ser Cys Glu Gly Leu Glu Arg Met Glu Arg Pro 50 55 60Glu Thr Tyr Lys Gln Trp Gln Val Arg Asn Gln Arg Val Gly Phe Lys 65 70 7580 Gln Leu Pro Leu Asn Gln Asp Met Met Lys Arg Ala Arg Xaa Glu Gly 85 9095 Gln Val Leu Pro Thr Arg Thr Phe Ile Ile Asp Glu Asp Asn Arg Trp 100105 110 Leu Leu Gln Gly Trp Lys Gly Arg Ile Leu Phe Ala Leu Ser Thr Trp115 120 125 Lys Pro Asp Asn Arg Ser Ser Ser Xaa 130 135 44 41 PRT Oryzasp. 44 Asn Gly Gly Ala Phe Ala Pro Ser Thr Trp Thr Ala Arg Ser Leu Asn 15 10 15 Gly Gly Ala Phe Ala Pro Ser Thr Trp Thr Ala Arg Ser Leu Pro Val20 25 30 Pro Ser Ser Pro Ser Thr Asp Ser Phe 35 40 45 1279 DNAArabidopsis thaliana 45 gcggctatct tctacggcca ccaccaccat acacctccgccggcaaagcg gctcaaccct 60 ggtcccgtgg ggataacaga gcagctggtt aaggcagcagaggtcataga gagcgacacg 120 tgtctagctc aggggatatt ggcgcggctc aatcaacagctctcttctcc cgtcgggaag 180 ccattagaaa gagcagcttt ttacttcaaa gaagctctcaataatctcct tcacaacgtc 240 tcccaaaccc taaaccctta ttccctcatc ttcaagatcgctgcttacaa atccttctca 300 gagatctctc ccgttcttca gttcgccaac tttacctccaaccaagccct cttagagtcc 360 ttccatggct tccaccgtct ccacatcatc gacttcgatatcggctacgg tggccaatgg 420 gcttccctca tgcaagagct tgttctccgc gacaacgccgctcctctctc cctcaagatc 480 accgttttcg cttctccggc gaaccacgac cagctcgaacttggcttcac tcaagacaac 540 ctcaagcact tcgcctctga gatcaacatc tcccttgacatccaagtttt gagcttagac 600 ctcctcggct ccatctcgtg gcctaactcg tcggagaaagaagctgtcgc cgttaacatc 660 tccgccgcgt ccttctcgca cctccctttg gtcctccgtttcgtgaagca tctatctccg 720 acgatcatcg tctgctccga cagaggatgc gagaggacggatctgccctt ctctcaacag 780 ctcgcccact cgctgcactc acacaccgct ctcttcgaatccctcgacgc cgtcaacgcc 840 aacctcgacg caatgcagaa gatcgagagg tttcttatacagccggagat agagaagctg 900 gtgttggatc gtagccgtcc gatagaaagg ccgatgatgacgtggcaagc gatgtttcta 960 cagatgggtt tctcaccggt gacgcacagt aacttcacggagtctcaagc cgagtgttta 1020 gtccaacgga cgccagtgag aggctttcac gtcgagaagaaacataactc acttctccta 1080 tgttggcaaa ggacagaact cgtcggagtt tcagcatggagatgtcgctc ctcctgattt 1140 ccaccggagt ttcaattatt aaaaaaatat tttccttaattcaatttatc ttaaatgaca 1200 aatttttagt ttctgatttt attttgctca gtgcgatggatttttaaatt taagtttcac 1260 acaaatatat aaatttttg 1279 46 379 PRTArabidopsis thaliana VARIANT (1)...(379) Xaa = Any Amino Acid 46 Ala AlaIle Phe Tyr Gly His His His His Thr Pro Pro Pro Ala Lys 1 5 10 15 ArgLeu Asn Pro Gly Pro Val Gly Ile Thr Glu Gln Leu Val Lys Ala 20 25 30 AlaGlu Val Ile Glu Ser Asp Thr Cys Leu Ala Gln Gly Ile Leu Ala 35 40 45 ArgLeu Asn Gln Gln Leu Ser Ser Pro Val Gly Lys Pro Leu Glu Arg 50 55 60 AlaAla Phe Tyr Phe Lys Glu Ala Leu Asn Asn Leu Leu His Asn Val 65 70 75 80Ser Gln Thr Leu Asn Pro Tyr Ser Leu Ile Phe Lys Ile Ala Ala Tyr 85 90 95Lys Ser Phe Ser Glu Ile Ser Pro Val Leu Gln Phe Ala Asn Phe Thr 100 105110 Ser Asn Gln Ala Leu Leu Glu Ser Phe His Gly Phe His Arg Leu His 115120 125 Ile Ile Asp Phe Asp Ile Gly Tyr Gly Gly Gln Trp Ala Ser Leu Met130 135 140 Gln Glu Leu Val Leu Arg Asp Asn Ala Ala Pro Leu Ser Leu LysIle 145 150 155 160 Thr Val Phe Ala Ser Pro Ala Asn His Asp Gln Leu GluLeu Gly Phe 165 170 175 Thr Gln Asp Asn Leu Lys His Phe Ala Ser Glu IleAsn Ile Ser Leu 180 185 190 Asp Ile Gln Val Leu Ser Leu Asp Leu Leu GlySer Ile Ser Trp Pro 195 200 205 Asn Ser Ser Glu Lys Glu Ala Val Ala ValAsn Ile Ser Ala Ala Ser 210 215 220 Phe Ser His Leu Pro Leu Val Leu ArgPhe Val Lys His Leu Ser Pro 225 230 235 240 Thr Ile Ile Val Cys Ser AspArg Gly Cys Glu Arg Thr Asp Leu Pro 245 250 255 Phe Ser Gln Gln Leu AlaHis Ser Leu His Ser His Thr Ala Leu Phe 260 265 270 Glu Ser Leu Asp AlaVal Asn Ala Asn Leu Asp Ala Met Gln Lys Ile 275 280 285 Glu Arg Phe LeuIle Gln Pro Glu Ile Glu Lys Leu Val Leu Asp Arg 290 295 300 Ser Arg ProIle Glu Arg Pro Met Met Thr Trp Gln Ala Met Phe Leu 305 310 315 320 GlnMet Gly Phe Ser Pro Val Thr His Ser Asn Phe Thr Glu Ser Gln 325 330 335Ala Glu Cys Leu Val Gln Arg Thr Pro Val Arg Gly Phe His Val Glu 340 345350 Lys Lys His Asn Ser Leu Leu Leu Cys Trp Gln Arg Thr Glu Leu Val 355360 365 Gly Val Ser Ala Trp Arg Cys Arg Ser Ser Xaa 370 375 47 745 DNAArabidopsis thaliana 47 tgcatacaac gcaccgtttt tcgtaacacg gtttcgcgaagctctatttc atttctcctc 60 gatttttgac atgcttgaga caattgtgcc acgagaagacgaagagagga tgttccttga 120 gatggaggtc tttgggagag aggcactgaa tgtgattgcttgcgaaggtt gggaaagagt 180 ggagaggcct gagacataca agcagtggca cgtacgggctatgaggtcag ggttggtgca 240 ggttccattt gacccaagca ttatgaagac atcgctgcataaggtccaca cattctacca 300 caaggatttt gtgatcgatc aagataaccg gtggctcttgcaaggctgga agggaagaac 360 tgtcatggct ctttctgttt ggaaaccaga gtccaaggcttgaccgagaa atcctcgttg 420 gcatatgaga gaccatctct tgattttctt cctgtgtaattcccagagac agaattacag 480 atgtaagaag agaatgctgc acaaagaact tgttcaaagataatattgat gtaagtcctg 540 ttttataact ttctagctgt gtttttgttg tttctcagctagattctcct aacggtattc 600 ttgtagctag ggtgatcaga ttgtttgtat attgctagcagagttagttt gtctagattg 660 taacacatat aagaggaagc ttagagtttc tatggtttaaagagaagttt tttccttctc 720 caatgtaaaa aaaaaaaaaa aaaaa 745 48 134 PRTArabidopsis thaliana VARIANT (1)...(134) Xaa = Any Amino Acid 48 Ala TyrAsn Ala Pro Phe Phe Val Thr Arg Phe Arg Glu Ala Leu Phe 1 5 10 15 HisPhe Ser Ser Ile Phe Asp Met Leu Glu Thr Ile Val Pro Arg Glu 20 25 30 AspGlu Glu Arg Met Phe Leu Glu Met Glu Val Phe Gly Arg Glu Ala 35 40 45 LeuAsn Val Ile Ala Cys Glu Gly Trp Glu Arg Val Glu Arg Pro Glu 50 55 60 ThrTyr Lys Gln Trp His Val Arg Ala Met Arg Ser Gly Leu Val Gln 65 70 75 80Val Pro Phe Asp Pro Ser Ile Met Lys Thr Ser Leu His Lys Val His 85 90 95Thr Phe Tyr His Lys Asp Phe Val Ile Asp Gln Asp Asn Arg Trp Leu 100 105110 Leu Gln Gly Trp Lys Gly Arg Thr Val Met Ala Leu Ser Val Trp Lys 115120 125 Pro Glu Ser Lys Ala Xaa 130 49 775 DNA Arabidopsis thaliana 49aaaaaatggg aaaccatcac tcttgatgaa cttatgatca atccaggaga gacaacggtc 60gtcaactgca ttcatcggtt acaatacact cctgatgaaa ctgtgtcatt agactctcca 120agagacacgg ttctgaagct attcagagat atcaatcctg acctctttgt gtttgcagag 180attaacggaa tgtacaactc tcctttcttc atgacgaggt tccgagaagc gctttttcat 240tactcttcac tctttgacat gtttgacacc acaatacacg cagaggatga gtacaaaaac 300aggtcactgt tggagagaga gttacttgtg agagacgcga tgagcgtgat ttcctgcgag 360ggtgcagagc ggtttgcgag gcctgaaacc tacaagcaat ggcgagttag gattttgaga 420gccgggttta agccagcaac tattagcaaa cagatcatga aggaggctaa ggaaattgtg 480aggaaacgtt accatagaga ttttgtgatc gatagcgata acaattggat gcttcaagga 540tggaaaggaa gagtcatcta tgctttttct tgctggaaac ctgctgagaa gttcacaaac 600aataatttaa acatctgaaa aatgttactt ctcaattaca tcatttttgt ttcccaatgg 660ttttgtagaa tatgtttgat cccgtgagtg gatgcaactc ttttttcctg caagtacata 720ttgtattcaa atccttgtgg aaatgataaa ttgtttaatc aaaaaaaaaa aaaaa 775 50 206PRT Arabidopsis thaliana VARIANT (1)...(206) Xaa = Any Amino Acid 50 LysLys Trp Glu Thr Ile Thr Leu Asp Glu Leu Met Ile Asn Pro Gly 1 5 10 15Glu Thr Thr Val Val Asn Cys Ile His Arg Leu Gln Tyr Thr Pro Asp 20 25 30Glu Thr Val Ser Leu Asp Ser Pro Arg Asp Thr Val Leu Lys Leu Phe 35 40 45Arg Asp Ile Asn Pro Asp Leu Phe Val Phe Ala Glu Ile Asn Gly Met 50 55 60Tyr Asn Ser Pro Phe Phe Met Thr Arg Phe Arg Glu Ala Leu Phe His 65 70 7580 Tyr Ser Ser Leu Phe Asp Met Phe Asp Thr Thr Ile His Ala Glu Asp 85 9095 Glu Tyr Lys Asn Arg Ser Leu Leu Glu Arg Glu Leu Leu Val Arg Asp 100105 110 Ala Met Ser Val Ile Ser Cys Glu Gly Ala Glu Arg Phe Ala Arg Pro115 120 125 Glu Thr Tyr Lys Gln Trp Arg Val Arg Ile Leu Arg Ala Gly PheLys 130 135 140 Pro Ala Thr Ile Ser Lys Gln Ile Met Lys Glu Ala Lys GluIle Val 145 150 155 160 Arg Lys Arg Tyr His Arg Asp Phe Val Ile Asp SerAsp Asn Asn Trp 165 170 175 Met Leu Gln Gly Trp Lys Gly Arg Val Ile TyrAla Phe Ser Cys Trp 180 185 190 Lys Pro Ala Glu Lys Phe Thr Asn Asn AsnLeu Asn Ile Xaa 195 200 205 51 548 DNA Arabidopsis thaliana 51aatcgcttga accgaatttg gatcgagatt cgaaagaaag gctgagagtg gagagagtgc 60tgttcggtag gaggattatg gatttggtcc gatcagatga tgataataat aaaccgggaa 120cccggtttgg gttaatggag gagaaagaac aatggagagt gttgatggag aaagctggat 180ttgagccggt taaaccgagt aattacgcgg ttagccaagc gaagctgcta ctatggaact 240acaattatag tacattgtat tcacttgttg aatcggagcc aggtttcatc tccttggctt 300ggaacaatgt gcctctcctc accgtttcct cttggcgttg actacttggt ccgataagtt 360aatctagtat tttgagttag cttttagaat tgaattgttt ggggttagat ttggatgttt 420aattagtctc tagcctattc tcttactctt ttttgtctag tgcttggagt gatgatggtt 480tgtcgtttat gttcatttgt aatatatatt gtatgtaaca tttgactaaa aaaaaaaaaa 540aaaaaaaa 548 52 113 PRT Arabidopsis thaliana VARIANT (1)...(113) Xaa =Any Amino Acid 52 Ser Leu Glu Pro Asn Leu Asp Arg Asp Ser Lys Glu ArgLeu Arg Val 1 5 10 15 Glu Arg Val Leu Phe Gly Arg Arg Ile Met Asp LeuVal Arg Ser Asp 20 25 30 Asp Asp Asn Asn Lys Pro Gly Thr Arg Phe Gly LeuMet Glu Glu Lys 35 40 45 Glu Gln Trp Arg Val Leu Met Glu Lys Ala Gly PheGlu Pro Val Lys 50 55 60 Pro Ser Asn Tyr Ala Val Ser Gln Ala Lys Leu LeuLeu Trp Asn Tyr 65 70 75 80 Asn Tyr Ser Thr Leu Tyr Ser Leu Val Glu SerGlu Pro Gly Phe Ile 85 90 95 Ser Leu Ala Trp Asn Asn Val Pro Leu Leu ThrVal Ser Ser Trp Arg 100 105 110 Xaa 53 1093 DNA Arabidopsis thaliana 53gcgaatgttg agatcttgga agcaatagct ggggaaacca gagtccacat tatcgatttt 60cagattgcac agggatcaca atacatgttt ttgattcagg agcttgcgaa acgccctggt 120gggccgccgt tgctgcgtgt gacgggtgtg gatgattcac agtccaccta tgctcgtggg 180ggaggactca gcttggtagg tgagaggctt gcaactttgg cgcagtcatg tggtgtcccg 240tttgagtttc acgatgccat catgtctggg tgcaaggtgc agcgggaaca tctcgggttg 300gaacctggct ttgctgttgt tgtgaacttc ccatatgtat tacaccacat gccagacgag 360agcgtaagtg ttgaaaaata cagagacagg ctgctgcatc tgatcaagag cctctcccca 420aaactggtta ctctagtaga gcaagaatcc aacacaaaca cctcgccatt ggtgtcacgg 480tttgtggaaa cactggatta ctacacagcg atgtttgagt cgatagatgc agcacggcca 540cgggatgata agcagagaat cagcgcagaa caacactgtg tagcaagaga catagtgaac 600atgatagcat gtgaggagtc agagagagta gagagacacg aggtactggg gaaatggagg 660gtcagaatga tgatggctgg gttcacgggt tggccggtca gcacatctgc agcgtttgca 720gcgagtgaga tgctgaaagc ttatgacaaa aactacaaac tgggaggcca tgaaggagcg 780ctctacctct tctggaagag acgacccatg gctacatgtt ccgtgtggaa gccaaaccca 840aactatattg ggtaagttat agtgatgatg gttacttgag tggataaaga agagcacaac 900aaaaacacat ctgtcgctgt aaatttttta ggatgtgcaa tgatgtttta agttgtaaca 960caacctaagt tatatatgta tacaaaccaa acctggtggt tgtttttctc ttgtaaattg 1020tcatgtggtt gtgggtggga agctagtaat gaaatataac caaaacattg attaggtcaa 1080aaaaaaaaaa aaa 1093 54 285 PRT Arabidopsis thaliana VARIANT (1)...(285)Xaa = Any Amino Acid 54 Ala Asn Val Glu Ile Leu Glu Ala Ile Ala Gly GluThr Arg Val His 1 5 10 15 Ile Ile Asp Phe Gln Ile Ala Gln Gly Ser GlnTyr Met Phe Leu Ile 20 25 30 Gln Glu Leu Ala Lys Arg Pro Gly Gly Pro ProLeu Leu Arg Val Thr 35 40 45 Gly Val Asp Asp Ser Gln Ser Thr Tyr Ala ArgGly Gly Gly Leu Ser 50 55 60 Leu Val Gly Glu Arg Leu Ala Thr Leu Ala GlnSer Cys Gly Val Pro 65 70 75 80 Phe Glu Phe His Asp Ala Ile Met Ser GlyCys Lys Val Gln Arg Glu 85 90 95 His Leu Gly Leu Glu Pro Gly Phe Ala ValVal Val Asn Phe Pro Tyr 100 105 110 Val Leu His His Met Pro Asp Glu SerVal Ser Val Glu Lys Tyr Arg 115 120 125 Asp Arg Leu Leu His Leu Ile LysSer Leu Ser Pro Lys Leu Val Thr 130 135 140 Leu Val Glu Gln Glu Ser AsnThr Asn Thr Ser Pro Leu Val Ser Arg 145 150 155 160 Phe Val Glu Thr LeuAsp Tyr Tyr Thr Ala Met Phe Glu Ser Ile Asp 165 170 175 Ala Ala Arg ProArg Asp Asp Lys Gln Arg Ile Ser Ala Glu Gln His 180 185 190 Cys Val AlaArg Asp Ile Val Asn Met Ile Ala Cys Glu Glu Ser Glu 195 200 205 Arg ValGlu Arg His Glu Val Leu Gly Lys Trp Arg Val Arg Met Met 210 215 220 MetAla Gly Phe Thr Gly Trp Pro Val Ser Thr Ser Ala Ala Phe Ala 225 230 235240 Ala Ser Glu Met Leu Lys Ala Tyr Asp Lys Asn Tyr Lys Leu Gly Gly 245250 255 His Glu Gly Ala Leu Tyr Leu Phe Trp Lys Arg Arg Pro Met Ala Thr260 265 270 Cys Ser Val Trp Lys Pro Asn Pro Asn Tyr Ile Gly Xaa 275 280285 55 1928 DNA Arabidopsis thaliana 55 aaagacttta gcagattttc aagcggctcagaacatcaac aacaacaaca acaacaaccg 60 ttttatagtc aagcagctct caacgcttttctttcaaggt ctgtgaagcc tcgaaattat 120 cagaattttc aatctccgtc ggccgatgattgatctcacg tcggtgaatg atatgagttt 180 gtttggtggt tctggttcat ctcagcgttacggtttaccg gttcccaggt ctcagacgca 240 acagcaacaa tcggattacg gtttatttggtgggatccga atgggaatcg ggtcgggtat 300 taataattat ccaacattaa ccggcgttccgtgtattgaa ccggttcaaa accgggttca 360 tgaatcggag aacatgttga atagtttaagagagcttgag aaacagcttt tagatgatga 420 cgatgagagt ggtggtgatg atgacgtgtcagttataaca aattcaaatt ccgattggat 480 tcaaaatctc gtgactccga acccgaacccgaacccggtt ttgtcttttt caccgagctc 540 ttcttcttcg tcttcttcgc cttctacagcttcgacgacg acatcggtat gttctaggca 600 aacggttatg gaaatcgcga cggcgatcgcggaagggaaa acagagatag cgacggagat 660 tttggcgcgt gtttctcaaa cgcctaatcttgagaggaat tcagaggaga agcttgttga 720 tttcatggtg gctgcgcttc gatcgaggatagcttctcca gtgacggaat tgtatgggaa 780 ggagcattta atctcgactc aattgctctacgagctctct ccttgtttca aactcggttt 840 cgaggccgcg aatctcgcca ttctcgacgccgccgataac aacgacggtg gaatgatgat 900 accgcacgtt atcgatttcg atatcggagaaggtggacaa tacgttaacc ttctccgtac 960 attatccacg cgccggaatg gtaaaagtcagagtcagaat tctccggtgg ttaagatcac 1020 cgccgtggcg aacaacgttt acggatgtttagtcgatgac ggtggagaag agaggttaaa 1080 agccgtcgga gatttgttga gccaactcggtgatcgactc ggtatctccg taagtttcaa 1140 cgtggtgacg agtttacgac tcggtgatctgaatcgtgaa tctctcgggt gtgatcccga 1200 cgagactttg gctgtgaact tagctttcaagctttatcgt gttcccgacg aaagcgtatg 1260 cacggagaat ccaagagacg aacttctccggcgcgtgaag ggacttaaac cgcgcgtggt 1320 tactctagtg gagcaagaaa tgaattcgaatacggcgccg tttttaggga gagtgagtga 1380 gtcatgcgcg tgttacggtg cgttgcttgagtcggtcgag tctacggttc ctagtacgaa 1440 ttccgaccgt gccaaagttg aggaaggaattggccggaag ctagtaaacg cggtggcgtg 1500 cgaaggaatc gatcgtatag agcggtgcgaggtgttcggg aaatggcgaa tgcggatgag 1560 catggctggg tttgagttaa tgccattgagtgagaagata gcggagtcga tgaagagtcg 1620 tggaaaccga gtccacccgg gctttaccgttaaagaagat aacggaggtg tgtgctttgg 1680 ttggatggga cgggcactca ctgtcgcatccgcttggcgt taacttcaca cactcttttt 1740 tttcttctta ttattaccat attattattaattttcgaga ttattctgat attattatca 1800 ttgtgatttt ccgtttcgaa aagtgtaggaatcttatgta acaaagaaaa aaaaaagact 1860 tttatgtttt tctaataata aaagaaagagtgattgggtt caaaaaaaaa aaaaaaaaaa 1920 aaaaaaaa 1928 56 524 PRTArabidopsis thaliana VARIANT (1)...(524) Xaa = Any Amino Acid 56 Asp LeuThr Ser Val Asn Asp Met Ser Leu Phe Gly Gly Ser Gly Ser 1 5 10 15 SerGln Arg Tyr Gly Leu Pro Val Pro Arg Ser Gln Thr Gln Gln Gln 20 25 30 GlnSer Asp Tyr Gly Leu Phe Gly Gly Ile Arg Met Gly Ile Gly Ser 35 40 45 GlyIle Asn Asn Tyr Pro Thr Leu Thr Gly Val Pro Cys Ile Glu Pro 50 55 60 ValGln Asn Arg Val His Glu Ser Glu Asn Met Leu Asn Ser Leu Arg 65 70 75 80Glu Leu Glu Lys Gln Leu Leu Asp Asp Asp Asp Glu Ser Gly Gly Asp 85 90 95Asp Asp Val Ser Val Ile Thr Asn Ser Asn Ser Asp Trp Ile Gln Asn 100 105110 Leu Val Thr Pro Asn Pro Asn Pro Asn Pro Val Leu Ser Phe Ser Pro 115120 125 Ser Ser Ser Ser Ser Ser Ser Ser Pro Ser Thr Ala Ser Thr Thr Thr130 135 140 Ser Val Cys Ser Arg Gln Thr Val Met Glu Ile Ala Thr Ala IleAla 145 150 155 160 Glu Gly Lys Thr Glu Ile Ala Thr Glu Ile Leu Ala ArgVal Ser Gln 165 170 175 Thr Pro Asn Leu Glu Arg Asn Ser Glu Glu Lys LeuVal Asp Phe Met 180 185 190 Val Ala Ala Leu Arg Ser Arg Ile Ala Ser ProVal Thr Glu Leu Tyr 195 200 205 Gly Lys Glu His Leu Ile Ser Thr Gln LeuLeu Tyr Glu Leu Ser Pro 210 215 220 Cys Phe Lys Leu Gly Phe Glu Ala AlaAsn Leu Ala Ile Leu Asp Ala 225 230 235 240 Ala Asp Asn Asn Asp Gly GlyMet Met Ile Pro His Val Ile Asp Phe 245 250 255 Asp Ile Gly Glu Gly GlyGln Tyr Val Asn Leu Leu Arg Thr Leu Ser 260 265 270 Thr Arg Arg Asn GlyLys Ser Gln Ser Gln Asn Ser Pro Val Val Lys 275 280 285 Ile Thr Ala ValAla Asn Asn Val Tyr Gly Cys Leu Val Asp Asp Gly 290 295 300 Gly Glu GluArg Leu Lys Ala Val Gly Asp Leu Leu Ser Gln Leu Gly 305 310 315 320 AspArg Leu Gly Ile Ser Val Ser Phe Asn Val Val Thr Ser Leu Arg 325 330 335Leu Gly Asp Leu Asn Arg Glu Ser Leu Gly Cys Asp Pro Asp Glu Thr 340 345350 Leu Ala Val Asn Leu Ala Phe Lys Leu Tyr Arg Val Pro Asp Glu Ser 355360 365 Val Cys Thr Glu Asn Pro Arg Asp Glu Leu Leu Arg Arg Val Lys Gly370 375 380 Leu Lys Pro Arg Val Val Thr Leu Val Glu Gln Glu Met Asn SerAsn 385 390 395 400 Thr Ala Pro Phe Leu Gly Arg Val Ser Glu Ser Cys AlaCys Tyr Gly 405 410 415 Ala Leu Leu Glu Ser Val Glu Ser Thr Val Pro SerThr Asn Ser Asp 420 425 430 Arg Ala Lys Val Glu Glu Gly Ile Gly Arg LysLeu Val Asn Ala Val 435 440 445 Ala Cys Glu Gly Ile Asp Arg Ile Glu ArgCys Glu Val Phe Gly Lys 450 455 460 Trp Arg Met Arg Met Ser Met Ala GlyPhe Glu Leu Met Pro Leu Ser 465 470 475 480 Glu Lys Ile Ala Glu Ser MetLys Ser Arg Gly Asn Arg Val His Pro 485 490 495 Gly Phe Thr Val Lys GluAsp Asn Gly Gly Val Cys Phe Gly Trp Met 500 505 510 Gly Arg Ala Leu ThrVal Ala Ser Ala Trp Arg Xaa 515 520 57 2635 DNA Arabidopsis thalianamisc_feature (1)...(2635) n = A,T,C or G 57 tcttactcaa ggttcttctttgtcatcttg ttgccgaatc cacaaagagg agaataaaga 60 ttcgaccttt attagatattaacgactctg gatttttggg tttttggagt tggatccaca 120 tgggttctta tccggatggattccctggat ccatggacga gttggatttc aataaggact 180 ttgatttgcc tccctcctcaaaccaaacct taggtttagc taatgggttc tatttagatg 240 acttagattt ctcatccttggatcctccag aggcatatcc ctcccagaac aacaacaaca 300 acaacatcaa caacaaagctgtagcaggag atctgttatc atcttcatct gatgacgctg 360 atttctctga ttctgttttgaagtatataa gccaagttct tatggaagag gatatggaag 420 agaagccttg tatgtttcatgatgctttgg ctcttcaagc tgctgagaaa tctctctatg 480 aggctcttgg tgagaaagacccttcttcgt cttctgcttc ttctgtggat catcctgaga 540 gattggctag tcatagccctgacggttctt gttcaggtgg tgcttttagt gattacgcta 600 gcaccactac cactacttcctctgattctc actggagtgt tgatggtttg gagaatagac 660 cttcttggtt acatacacctatgccgagta attttgtttt ccagtctact tctaggtcca 720 acagtgtcac cggtggtggtggtggtggta atagtgcggt ttacggttca ggttttggcg 780 atgatttggt ttcgaatatgtttaaagatg atgaattggc tatgcagttc aagaaagggg 840 ttgaggaagc tagtaagttccttcctaagt cttctcagct ctttattgat gtggatagtt 900 acatccctat gaattctggttccaaggaaa atggttctga ggtttttgtt aagacggaga 960 agaaagatga gacagagcatcatcatcatc atagctatgc accaccaccc aacagattaa 1020 ctggtaagaa aagccattggcgcgacgaag atgaagattt cgttgaagaa agaagtaaca 1080 agcaatcagc tgtttatgttgaggaaagcg agctttctga aatgtttgat aacatgttcc 1140 tatgtggccc tgggaaacctgtatgcattc ttaaccagaa ctttcctaca gaatccgcta 1200 aagtcgtgac cgcacagtcaaatggagcaa agattcgtgg gaagaaatca acttctacta 1260 gtcatagtaa cgattctaagaaagaaactg ctgatttgag gactcttttg gtgttatgtg 1320 cacaagctgt atcagtggatgatcgtagaa ccgccaacgt ttagctaagg cagatacgag 1380 agcattcttc gcctctaggcaatggttcag agcggttggc tcattatttt gcaaatagtc 1440 ttgaagcacg cttagctgggaccggtacac agatctacac cgctttatct tcgaagaaaa 1500 cgtctgcagc agacatgttgaaggcttacc agacatacat gtcggtctgc cctttcaaga 1560 aagctgctat catatttgctaaccacagca tgatgcgttt cactgcaaac gccaacacga 1620 tccacataat agatttcggaatatcttacg gttttcagtg gcctgctctg attcatcgcc 1680 tctcgctcag cagacctggtggttcgccta agcttcgaat taccggtnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnnnnngagttca ggagacaggt catcgcttgg ctcgatactg 1800 tcagcgacac aatgttccgtttgagtacaa cgcaattgct cagaaatggg gaaacgatcc 1860 aagtcgaaga cttaaagcttcgacaaggag agtatgtggt tgtgaactct ttgttccgtt 1920 tcaggaacct tctagatgagaccgttctgg taaacagccc gagagatgca gttttgaagc 1980 tgataagaaa aataaacccgaatgtcttca ttccagcgat cttaagcggg aattacaacg 2040 cgccattctt tgtcacgaggttcagagaag cgttgtttca ttactcggct gtgtttgata 2100 tgtgtgactc gaagctagctagggaagacg agatgaggct gatgtatgtg tttgagtttt 2160 atgggagaga gattgtgaatgttgtggctt ctgaaggaac agagagagtg gagagccgag 2220 agacatataa gcagtggcaggcgagactga tccgagccgg atttagacag cttccgcttg 2280 agaaggaact gatgcagaatctgaagttga aaatcgaaaa cgggtacgat aaaaacttcg 2340 atgttgatca aaacggtaactggttacttc aagggtggaa aggtagaatc gtgtatgctt 2400 catctctatg ggttccttcgtcttcataga tgttgtttct tacgttctaa gcgactggga 2460 tttatgtagg gcttttctgttgatagtctc tcgccaacac gagtggatta agttcagagt 2520 tagggttctt gaacactagaatgttgttat attatgcttg tgacatagcg tgtgtaagag 2580 tgtagcctaa gagatatagtactcattgca tgatcttttg ctatatgttn catgt 2635 58 809 PRT Arabidopsisthaliana VARIANT (1)...(809) Xaa = Any Amino Acid 58 Leu Leu Lys Val LeuLeu Cys His Leu Val Ala Glu Ser Thr Lys Arg 1 5 10 15 Arg Ile Lys IleArg Pro Leu Leu Asp Ile Asn Asp Ser Gly Phe Leu 20 25 30 Gly Phe Trp SerTrp Ile His Met Gly Ser Tyr Pro Asp Gly Phe Pro 35 40 45 Gly Ser Met AspGlu Leu Asp Phe Asn Lys Asp Phe Asp Leu Pro Pro 50 55 60 Ser Ser Asn GlnThr Leu Gly Leu Ala Asn Gly Phe Tyr Leu Asp Asp 65 70 75 80 Leu Asp PheSer Ser Leu Asp Pro Pro Glu Ala Tyr Pro Ser Gln Asn 85 90 95 Asn Asn AsnAsn Asn Ile Asn Asn Lys Ala Val Ala Gly Asp Leu Leu 100 105 110 Ser SerSer Ser Asp Asp Ala Asp Phe Ser Asp Ser Val Leu Lys Tyr 115 120 125 IleSer Gln Val Leu Met Glu Glu Asp Met Glu Glu Lys Pro Cys Met 130 135 140Phe His Asp Ala Leu Ala Leu Gln Ala Ala Glu Lys Ser Leu Tyr Glu 145 150155 160 Ala Leu Gly Glu Lys Asp Pro Ser Ser Ser Ser Ala Ser Ser Val Asp165 170 175 His Pro Glu Arg Leu Ala Ser His Ser Pro Asp Gly Ser Cys SerGly 180 185 190 Gly Ala Phe Ser Asp Tyr Ala Ser Thr Thr Thr Thr Thr SerSer Asp 195 200 205 Ser His Trp Ser Val Asp Gly Leu Glu Asn Arg Pro SerTrp Leu His 210 215 220 Thr Pro Met Pro Ser Asn Phe Val Phe Gln Ser ThrSer Arg Ser Asn 225 230 235 240 Ser Val Thr Gly Gly Gly Gly Gly Gly AsnSer Ala Val Tyr Gly Ser 245 250 255 Gly Phe Gly Asp Asp Leu Val Ser AsnMet Phe Lys Asp Asp Glu Leu 260 265 270 Ala Met Gln Phe Lys Lys Gly ValGlu Glu Ala Ser Lys Phe Leu Pro 275 280 285 Lys Ser Ser Gln Leu Phe IleAsp Val Asp Ser Tyr Ile Pro Met Asn 290 295 300 Ser Gly Ser Lys Glu AsnGly Ser Glu Val Phe Val Lys Thr Glu Lys 305 310 315 320 Lys Asp Glu ThrGlu His His His His His Ser Tyr Ala Pro Pro Pro 325 330 335 Asn Arg LeuThr Gly Lys Lys Ser His Trp Arg Asp Glu Asp Glu Asp 340 345 350 Phe ValGlu Glu Arg Ser Asn Lys Gln Ser Ala Val Tyr Val Glu Glu 355 360 365 SerGlu Leu Ser Glu Met Phe Asp Asn Met Phe Leu Cys Gly Pro Gly 370 375 380Lys Pro Val Cys Ile Leu Asn Gln Asn Phe Pro Thr Glu Ser Ala Lys 385 390395 400 Val Val Thr Ala Gln Ser Asn Gly Ala Lys Ile Arg Gly Lys Lys Ser405 410 415 Thr Ser Thr Ser His Ser Asn Asp Ser Lys Lys Glu Thr Ala AspLeu 420 425 430 Arg Thr Leu Leu Val Leu Cys Ala Gln Ala Val Ser Val AspAsp Arg 435 440 445 Arg Thr Ala Asn Val Xaa Leu Arg Gln Ile Arg Glu HisSer Ser Pro 450 455 460 Leu Gly Asn Gly Ser Glu Arg Leu Ala His Tyr PheAla Asn Ser Leu 465 470 475 480 Glu Ala Arg Leu Ala Gly Thr Gly Thr GlnIle Tyr Thr Ala Leu Ser 485 490 495 Ser Lys Lys Thr Ser Ala Ala Asp MetLeu Lys Ala Tyr Gln Thr Tyr 500 505 510 Met Ser Val Cys Pro Phe Lys LysAla Ala Ile Ile Phe Ala Asn His 515 520 525 Ser Met Met Arg Phe Thr AlaAsn Ala Asn Thr Ile His Ile Ile Asp 530 535 540 Phe Gly Ile Ser Tyr GlyPhe Gln Trp Pro Ala Leu Ile His Arg Leu 545 550 555 560 Ser Leu Ser ArgPro Gly Gly Ser Pro Lys Leu Arg Ile Thr Gly Xaa 565 570 575 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Phe Arg Arg Gln 580 585 590 Val IleAla Trp Leu Asp Thr Val Ser Asp Thr Met Phe Arg Leu Ser 595 600 605 ThrThr Gln Leu Leu Arg Asn Gly Glu Thr Ile Gln Val Glu Asp Leu 610 615 620Lys Leu Arg Gln Gly Glu Tyr Val Val Val Asn Ser Leu Phe Arg Phe 625 630635 640 Arg Asn Leu Leu Asp Glu Thr Val Leu Val Asn Ser Pro Arg Asp Ala645 650 655 Val Leu Lys Leu Ile Arg Lys Ile Asn Pro Asn Val Phe Ile ProAla 660 665 670 Ile Leu Ser Gly Asn Tyr Asn Ala Pro Phe Phe Val Thr ArgPhe Arg 675 680 685 Glu Ala Leu Phe His Tyr Ser Ala Val Phe Asp Met CysAsp Ser Lys 690 695 700 Leu Ala Arg Glu Asp Glu Met Arg Leu Met Tyr ValPhe Glu Phe Tyr 705 710 715 720 Gly Arg Glu Ile Val Asn Val Val Ala SerGlu Gly Thr Glu Arg Val 725 730 735 Glu Ser Arg Glu Thr Tyr Lys Gln TrpGln Ala Arg Leu Ile Arg Ala 740 745 750 Gly Phe Arg Gln Leu Pro Leu GluLys Glu Leu Met Gln Asn Leu Lys 755 760 765 Leu Lys Ile Glu Asn Gly TyrAsp Lys Asn Phe Asp Val Asp Gln Asn 770 775 780 Gly Asn Trp Leu Leu GlnGly Trp Lys Gly Arg Ile Val Tyr Ala Ser 785 790 795 800 Ser Leu Trp ValPro Ser Ser Ser Xaa 805 59 90 PRT Oryza sp. VARIANT (1)...(90) Xaa = AnyAmino Acid 59 Gln Glu Ala Asp His Asn Lys Thr Gly Phe Leu Asp Arg PheThr Glu 1 5 10 15 Ala Leu Phe Tyr Tyr Ser Ala Val Phe Asp Ser Leu AspAla Ala Asn 20 25 30 Asn Asn Asn Asn Asn Asn Asn Gln Arg Met Glu Ala GluTyr Leu Gln 35 40 45 Arg Glu Ile Cys Asp Ile Val Cys Gly Glu Gly Ala AlaArg Xaa Glu 50 55 60 Arg His Glu Pro Leu Ser Arg Trp Arg Asp Arg Leu ThrArg Ala Gly 65 70 75 80 Leu Ser Ala Val Pro Leu Gly Ser Asn Ala 85 90 60199 DNA Daucus carota misc_feature (1)...(199) n = A,T,C or G 60tctgcagaca attttnagga ggccaatacc atgctattgg aaatttcaga actgtccaca 60cctnnnnnnn nnnnnnnnnn nnnnnnnnnn nnngtacttc tcagaggnaa tgtcggnnag 120attagttagc tcctgcttag gaatctatgc ttctcttccn gcaacagtgg tgcctcctca 180tggtcagaaa gtggcctca 199 61 66 PRT Daucus carota VARIANT (1)...(66) Xaa= Any Amino Acid 61 Ser Ala Asp Asn Phe Xaa Glu Ala Asn Thr Met Leu LeuGlu Ile Ser 1 5 10 15 Glu Leu Ser Thr Pro Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Tyr 20 25 30 Phe Ser Glu Xaa Met Ser Xaa Arg Leu Val Ser SerCys Leu Gly Ile 35 40 45 Tyr Ala Ser Leu Pro Ala Thr Val Val Pro Pro HisGly Gln Lys Val 50 55 60 Ala Ser 65 62 321 DNA Glycine max misc_feature(1)...(321) n = A,T,C or G 62 tcaactgaga atctagaaga tgccaacaagatgcttctgg agatttctca gttatcaaca 60 ccgttcnnca cttcagcaca gcgtgtggcagcatatttct cagaagccat atcagcaagg 120 ttggtgagtt catgtctagg gatatacgcaactttgccac acacacacca aagccacaag 180 gtagcttcag cttttcaagt gttcaatggtattagtcctt tagtggagtt ctcacacttc 240 acagcaaacc aagcaattca agaagccttcgaaagagaag agagggtgca catcatagat 300 cttgatataa tgcaagggtt g 321 63 107PRT Glycine max VARIANT (1)...(107) Xaa = Any Amino Acid 63 Ser Thr GluAsn Leu Glu Asp Ala Asn Lys Met Leu Leu Glu Ile Ser 1 5 10 15 Gln LeuSer Thr Pro Phe Xaa Thr Ser Ala Gln Arg Val Ala Ala Tyr 20 25 30 Phe SerGlu Ala Ile Ser Ala Arg Leu Val Ser Ser Cys Leu Gly Ile 35 40 45 Tyr AlaThr Leu Pro His Thr His Gln Ser His Lys Val Ala Ser Ala 50 55 60 Phe GlnVal Phe Asn Gly Ile Ser Pro Leu Val Glu Phe Ser His Phe 65 70 75 80 ThrAla Asn Gln Ala Ile Gln Glu Ala Phe Glu Arg Glu Glu Arg Val 85 90 95 HisIle Ile Asp Leu Asp Ile Met Gln Gly Leu 100 105 64 195 DNA Picea abiesmisc_feature (1)...(195) n = A,T,C or G 64 tctgcagaca actttgaagaagccaataca atactgcctc agatcacaga actctccacc 60 ccctatngca actcggtgcaacgagtggct gcctatnnnn nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn nntgcataggaatgtattct cctctccctc ctattcacat gtcccagagc 180 cagaaaattg tgaat 195 6565 PRT Picea abies VARIANT (1)...(65) Xaa = Any Amino Acid 65 Ser AlaAsp Asn Phe Glu Glu Ala Asn Thr Ile Leu Pro Gln Ile Thr 1 5 10 15 GluLeu Ser Thr Pro Tyr Xaa Asn Ser Val Gln Arg Val Ala Ala Tyr 20 25 30 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Ile Gly Met 35 40 45 TyrSer Pro Leu Pro Pro Ile His Met Ser Gln Ser Gln Lys Ile Val 50 55 60 Asn65 66 2151 DNA Zea mays 66 gatatcagca tcatcaattt taaatgtaag ttggcaaaagatcatgaggg ttctcatagt 60 aatttggcca caaggtatga cactgtctca attgagcaatctagtagaga aactgatcca 120 tcatatattg ctcatattga aagtgaaaaa gatatgctcaagaacctagt agagaagcta 180 aaaattgaaa aatctagctc tactagaaaa atatgataggttgcctgttt ctcatgaaaa 240 tttattagat aatcatatca tggctagatg tcgctcatgaggttgttctt gctagtttag 300 attcctgtgg gcattcatct cttttagatg cactaacatgataggaagtt tctaatctgg 360 tgcttcacaa ttctggtgat tcatgcttcc ttcattgcaattgatattga tgcttgattc 420 atgcttcagt cactttgtgc gtttaattgg tattgtatgtatcactagat tgtagggtgt 480 ctgcaactag tgtttcacca tgtggttttt tagtatcattcgtattagtt tctaactttc 540 tattgatata ttaaagtgat aactagtttt agaaatattctcttgtgcca ttaatgctac 600 aacttgtttt tagcgtgtac gttagcatta taatatttccttattatgaa agcggaagag 660 aaacgcgccc aaccagagca tccacgtcgt ctcatttcaccttcatcgtt ggatcataga 720 tgagcggtcc acggtgaact ccgtttgcct gcaaaaccacgtcctctacg cgctgttaag 780 tagcttctag aaacatcacg atgtgtcccg tccattcctttaggaggagc cggatccggc 840 gccgcagtcg cccaaggtcc cgaccgccgc ggcctcggccgccgccgcca aggagcggaa 900 ggaggtgcag cggcggaagc agcgcgacga ggagggcctccacctgctga gtgctgacgc 960 tgctgctgca gtgcgcggag gccgtgaacg cggacaacctcgacgacgcg caccagacgc 1020 tgctggagat cgcggagctg gccacgccgt tcggcacctcgacccagcgc gtggccgcct 1080 acttcgcgga ggccatgtcg gcgcgcgtcg tcagctcctgcctaggcctg tacgcgccgc 1140 tgccgccggg ctcccccgcc gcggcgcgcc tccacggccgcgtggccgcc gcgttccagg 1200 tgttcaacgg catcagcccc ttcgtcaagt tctcgcacttcaccgccaac caggccatcc 1260 aggaggcgtt cgagcgggag gagcgtgtgc acatcatcgacctcgacatc atgcaggggc 1320 tgcagtggcc gggcctcttc cacatccttg tctcccgccccggcggcccg cccagggtca 1380 ggctcaccgg cctgggggcg tccatggacg cgctcgaggcgacggggaag cgcctctccg 1440 acttcgccga cacgctcggc ctgcccttcg agttctgcgccgtcgccgag aaggccggca 1500 acgttgaccc gcagaagctg ggcgtcacgc ggcgggaggccgtcgccgtc cactggccgc 1560 accactcgct ttacgacgtc atcggctccg actccaacacgctctggctc atccaaaggt 1620 cctccatttt ccttctctgc ctttcttcca tgtcaaatcttgatgcaatc atgaccactt 1680 ttcagctgct gacattggat aatgtgagct ttacggcaagcatcaagtcg tggtagtaca 1740 tccattacag ctatttctaa aatattcttc ggaggtttcctgctcatagt aaaaaaaaat 1800 cgcgttttga agctcaaaag gcgatttctt ccgaggtttgctgttgagcg ctattttgga 1860 aaccccattt tctcaattga tttttatttt ttaaagaaaaattagttcat ttttctcttg 1920 tgaaatggag tcccaaacta accctaatat taaaaaaaacgcgctttgga gctcaaaacg 1980 ctcgttgtta tgaccaacca gctttatagg tttaaaaaggttgaatcttg acaatgcttt 2040 tgaaaaggtt gaatcttgac aatgcttttg agatgatactgtagtgtagt ctgtagtgga 2100 gcatcctcca tggtctttgg tgatcgagaa ttcctgcagcccgggggatc c 2151 67 716 PRT Zea mays VARIANT (1)...(716) Xaa = AnyAmino Acid 67 Tyr Gln His His Gln Phe Xaa Met Xaa Val Gly Lys Arg SerXaa Gly 1 5 10 15 Phe Ser Xaa Xaa Phe Gly His Lys Val Xaa His Cys LeuAsn Xaa Ala 20 25 30 Ile Xaa Xaa Arg Asn Xaa Ser Ile Ile Tyr Cys Ser TyrXaa Lys Xaa 35 40 45 Lys Arg Tyr Ala Gln Glu Pro Ser Arg Glu Ala Lys AsnXaa Lys Ile 50 55 60 Xaa Leu Tyr Xaa Lys Asn Met Ile Gly Cys Leu Phe LeuMet Lys Ile 65 70 75 80 Tyr Xaa Ile Ile Ile Ser Trp Leu Asp Val Ala HisGlu Val Val Leu 85 90 95 Ala Ser Leu Asp Ser Cys Gly His Ser Ser Leu LeuAsp Ala Leu Thr 100 105 110 Xaa Xaa Glu Val Ser Asn Leu Val Leu His AsnSer Gly Asp Ser Cys 115 120 125 Phe Leu His Cys Asn Xaa Tyr Xaa Cys LeuIle His Ala Ser Val Thr 130 135 140 Leu Cys Val Xaa Leu Val Leu Tyr ValSer Leu Asp Cys Arg Val Ser 145 150 155 160 Ala Thr Ser Val Ser Pro CysGly Phe Leu Val Ser Phe Val Leu Val 165 170 175 Ser Asn Phe Leu Leu IleTyr Xaa Ser Asp Asn Xaa Phe Xaa Lys Tyr 180 185 190 Ser Leu Val Pro LeuMet Leu Gln Leu Val Phe Ser Val Tyr Val Ser 195 200 205 Ile Ile Ile PhePro Tyr Tyr Glu Ser Gly Arg Glu Thr Arg Pro Thr 210 215 220 Arg Ala SerThr Ser Ser His Phe Thr Phe Ile Val Gly Ser Xaa Met 225 230 235 240 SerGly Pro Arg Xaa Thr Pro Phe Ala Cys Lys Thr Thr Ser Ser Thr 245 250 255Arg Cys Xaa Val Ala Ser Arg Asn Ile Thr Met Cys Pro Val His Ser 260 265270 Phe Arg Arg Ser Arg Ile Arg Arg Arg Ser Arg Pro Arg Ser Arg Pro 275280 285 Pro Arg Pro Arg Pro Pro Pro Pro Arg Ser Gly Arg Arg Cys Ser Gly290 295 300 Gly Ser Ser Ala Thr Arg Arg Ala Ser Thr Cys Xaa Val Leu ThrLeu 305 310 315 320 Leu Leu Gln Cys Ala Glu Ala Val Asn Ala Asp Asn LeuAsp Asp Ala 325 330 335 His Gln Thr Leu Leu Glu Ile Ala Glu Leu Ala ThrPro Phe Gly Thr 340 345 350 Ser Thr Gln Arg Val Ala Ala Tyr Phe Ala GluAla Met Ser Ala Arg 355 360 365 Val Val Ser Ser Cys Leu Gly Leu Tyr AlaPro Leu Pro Pro Gly Ser 370 375 380 Pro Ala Ala Ala Arg Leu His Gly ArgVal Ala Ala Ala Phe Gln Val 385 390 395 400 Phe Asn Gly Ile Ser Pro PheVal Lys Phe Ser His Phe Thr Ala Asn 405 410 415 Gln Ala Ile Gln Glu AlaPhe Glu Arg Glu Glu Arg Val His Ile Ile 420 425 430 Asp Leu Asp Ile MetGln Gly Leu Gln Trp Pro Gly Leu Phe His Ile 435 440 445 Leu Val Ser ArgPro Gly Gly Pro Pro Arg Val Arg Leu Thr Gly Leu 450 455 460 Gly Ala SerMet Asp Ala Leu Glu Ala Thr Gly Lys Arg Leu Ser Asp 465 470 475 480 PheAla Asp Thr Leu Gly Leu Pro Phe Glu Phe Cys Ala Val Ala Glu 485 490 495Lys Ala Gly Asn Val Asp Pro Gln Lys Leu Gly Val Thr Arg Arg Glu 500 505510 Ala Val Ala Val His Trp Pro His His Ser Leu Tyr Asp Val Ile Gly 515520 525 Ser Asp Ser Asn Thr Leu Trp Leu Ile Gln Arg Ser Ser Ile Phe Leu530 535 540 Leu Cys Leu Ser Ser Met Ser Asn Leu Asp Ala Ile Met Thr ThrPhe 545 550 555 560 Gln Leu Leu Thr Leu Asp Asn Val Ser Phe Thr Ala SerIle Lys Ser 565 570 575 Trp Xaa Tyr Ile His Tyr Ser Tyr Phe Xaa Asn IleLeu Arg Arg Phe 580 585 590 Pro Ala His Ser Lys Lys Lys Ser Arg Phe GluAla Gln Lys Ala Ile 595 600 605 Ser Ser Glu Val Cys Cys Xaa Ala Leu PheTrp Lys Pro His Phe Leu 610 615 620 Asn Xaa Phe Leu Phe Phe Lys Glu LysLeu Val His Phe Ser Leu Val 625 630 635 640 Lys Trp Ser Pro Lys Leu ThrLeu Ile Leu Lys Lys Thr Arg Phe Gly 645 650 655 Ala Gln Asn Ala Arg CysTyr Asp Gln Pro Ala Leu Xaa Val Xaa Lys 660 665 670 Gly Xaa Ile Leu ThrMet Leu Leu Lys Arg Leu Asn Leu Asp Asn Ala 675 680 685 Phe Glu Met IleLeu Xaa Cys Ser Leu Xaa Trp Ser Ile Leu His Gly 690 695 700 Leu Trp XaaSer Arg Ile Pro Ala Ala Arg Gly Ile 705 710 715 68 23 DNA ArtificialSequence CDS (1)...(23) Primer 68 cay tty acn gcn aay car gcn at 23 69 8PRT Artificial Sequence primer 69 His Phe Thr Ala Asn Gln Ala Ile 1 5 7029 DNA Artificial Sequence CDS (10)...(29) Primer 70 acgtctcga gtn cayath ath gay ttn ga 29 71 7 PRT Artificial Sequence VARIANT (1)...(7) Xaa= Any Amino Acid 71 Val His Ile Ile Asp Xaa Asp 1 5 72 20 DNA ArtificialSequence CDS (1)...(20) Primer 72 ytn car tgy gcn gar gcn gt 20 73 7 PRTArtificial Sequence Primer 73 Leu Gln Cys Ala Glu Ala Val 1 5 74 23 DNAArtificial Sequence CDS (3)...(23) Primer 74 ck ccm gtk tgg ngg ncc nccngg 23 75 8 PRT Artificial Sequence VARIANT (1)...(8) Xaa = Any AminoAcid 75 Pro Gly Gly Pro Pro Xaa Xaa Arg 1 5 76 23 DNA ArtificialSequence CDS (3)...(23) Primer 76 at ncc rtt raa nac ytg raa ngc 23 77 8PRT Artificial Sequence Primer 77 Ala Phe Gln Val Phe Asn Gly Ile 1 5 7823 DNA Artificial Sequence CDS (3)...(23) Primer 78 at rtg raa nar nccngg cca ytg 23 79 8 PRT Artificial Sequence Primer 79 Gln Trp Pro GlyLeu Phe His Ile 1 5

What is claimed is:
 1. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a SCARECROW protein containing an amino acid sequence substantially similar to the sequence of MOTIF III (VHIID) of Arabidopsis SCR protein shown in FIGS. 13A-F.
 2. An isolated nucleic acid molecule comprising (a) a nucleotide sequence that encodes a scarecrow protein having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67; or (b) the complement of the nucleotide sequence of (a).
 3. An isolated nucleic acid molecule comprising a nucleotide sequence that hybridizes to the nucleic acid of claim 2 and encodes a naturally occurring SCR gene product.
 4. A nucleic acid molecule comprising (a) a nucleotide sequence that encodes a SCR protein lacking one to four of the following motifs delineated in FIGS. 13A-F: MOTIF I, MOTIF II, MOTIF III, MOTIF IV, MOTIF V, or MOTIF VI; or (b) the complement of the nucleotide sequence of (a).
 5. A nucleic acid molecule comprising (a) a nucleotide sequence that encodes a polypeptide corresponding to MOTIF I, MOTIF II, MOTIF IV, MOTIF V or MOTIF VI of the SCARECROW protein delineated in FIGS. 13A-F; or (b) the complement of the nucleotide sequence of (a).
 6. The isolated nucleic acid molecule of claim 1 comprising the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64 or SEQ ID NO:66.
 7. A DNA vector containing the nucleic acid molecule of claim 1, 2, 3, 4, 5, or
 6. 8. An expression vector containing the nucleic acid molecule of claim 1, 2, 3, 4, 5, or 6 operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the nucleotide sequence in a host cell.
 9. A genetically-engineered host cell containing the nucleic acid molecule of claim 1, 2, 3, 4, 5, or
 6. 10. A genetically-engineered host cell containing the nucleic acid molecule of claim 1, 2, 3, 4, 5, or 6 operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the nucleotide sequence in a host cell.
 11. An isolated SCARECROW protein.
 12. The protein of claim 11 having the amino acid sequence shown in FIG. 5E (SEQ ID NO:2).
 13. A SCARECROW protein lacking one to four of the following motifs delineated in FIGS. 13A-F: MOTIF I, MOTIF II, MOTIF III, MOTIF VI, MOTIF V, or MOTIF VI.
 14. A polypeptide corresponding to MOTIF I, MOTIF II, MOTIF IV, MOTIF V or MOTIF VI of the SCARECROW protein as delineated in FIGS. 13A-F.
 15. An antibody that immunospecifically binds the protein or polypeptide of claim 11, 12, 13 or
 14. 16. An anti-idiotypic antibody that mimics an epitope of SCARECROW protein.
 17. A plant genetically-engineered to overexpress or underexpress a SCARECROW protein or polypeptide, so that cell division is modified, and root and/or stem development is altered.
 18. A plant genetically-engineered to overexpress a SCARECROW protein or polypeptide, so that cell division is increased in roots, resulting in thicker root development.
 19. A transgenic plant containing a transgene having the nucleic acid molecule of claim 1, 2, 3, 4, 5, or
 6. 20. A transgenic plant containing a transgene having the nucleic acid molecule of claim 1, 2, 3, 4, 5, or 6 operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the nucleotide sequence in a transgenic plant cell.
 21. The transgenic plant of claim 19, in which the transgene encodes an antisense nucleotide sequence that suppresses expression of endogenous SCARECROW gene product, so that cell division is decreased in roots, resulting in thinner root development.
 22. A genetically-engineered plant in which the endogenous SCARECROW gene is disrupted or inactivated so that cell division is decreased in roots, resulting in thinner root development.
 23. A transgenic plant containing a transgene encoding a gene of interest operatively associated with a SCARECROW promoter, so that the gene of interest is expressed in roots.
 24. The transgenic plant of claim 23, in which the gene of interest encodes a gene product that confers herbicide, salt, pathogen, or insect resistance.
 25. A transgenic plant containing a transgene encoding a gene of interest operatively associated with a SCARECROW promoter, so that the gene of interest is expressed in stems.
 26. The transgenic plant of claim 25, in which the gene of interest encodes a gene product that increases starch, lignin or cellulose biosynthesis.
 27. A plant genetically-engineered to overexpress or underexpress the SCARECROW protein so that gravitropism of the stem or hypocotyl is altered.
 28. The plant of claim 27, which is less susceptible to lodging than a wild-type plant. 